A SYSTEMATIC APPROACH TO BACTERIAL IDENTIFICATION

This is a practical guide for microbiologists on screening products for objectionable microorganisms, profiling environmental microbiota, and investigating contamination events to determine a likely source of origin. Microbial identification is a critical component of the microbiology function.

NEED TO IDENTIFY 

In the pharmaceutical, healthcare and cosmetic manufacturing industries, we identify and classify micro-organisms for various reasons, such as:

• To identify the source of contamination and implement preventive actions to reduce the risk of recurrence in the future.
• To assess the microbial flora associated with the area and the process.
• To monitor and analyze trends in microbial contamination.
• To determine the microbiological risk to the product, the patient, and the consumer.
• To establish a reference library of organisms for culture media.  

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HISTORY OF MICROBIAL IDENTIFICATION 

In the early stages of microbial identification, isolates were classified using basic phenotypic characteristics, such as colony morphology and cellular morphology.

Taxonomy is the scientific discipline that classifies microorganisms based on shared morphological, physiological, and genetic characteristics and assigns them appropriate names.

A major milestone in microbial identification occurred in 1884, when Dr Hans Christian Gram developed a staining technique that enhanced the visibility of bacterial cells under the microscope.

This method is widely used to differentiate microorganisms into Gram-positive and Gram-negative types.

• Phenotypic methods - These are based on morphological, physiological and/ or biochemical characteristics expressed by the organism during growth.
• Chemotaxonomic methods - These are based on the chemical constituents of the cell, for example, antigen content, cell wall composition, cellular fatty acid content or whole cell protein content.
• Genotypic methods – These are based on the genetic makeup of the organism, for example, its nucleic acids.

There is no single universal method capable of identifying all microorganisms; therefore, a polyphasic approach that combines several identification methods is often applied, particularly when a definitive identification is required.

However, it is important to recognise that no method is 100% accurate, and sufficient supporting data must be obtained for each isolate to ensure confidence in identification, regardless of the method used.

PHENOTYPIC MICROBIAL IDENTIFICATION METHODS

Phenotypical identification is based on an organism's morphology, metabolic attributes or chemical profile. 

The following phenotypic characteristics can be used to identify a microorganism: 

1. Microscopic Morphology: 

Microscopic examination generally provides useful information about the isolate recovered from a product, material, or the environment. A simple wet preparation of the bacterial colony examined under oil immersion can also provide clues to its possible identity, depending on its size, shape, arrangements and motility. Although basic, this information is fundamental in determining the appropriate next steps or further identification.

Bacterial spore stain: 

Endospore formation is an important physiological survival characteristic of certain Gram-positive rods, such as species of Bacillus and Clostridium.

Spore staining using malachite green and safranin counterstain is a useful technique for determining the presence and absence of spores and for identifying their location within the mother cell (for example, terminal or sub-terminal), which can serve as a distinguishing feature for some spore-forming organisms. This is referred to as the Schaeffer-Fulton stain.

1. Colony Morphology:

Colony morphology on solid media can provide valuable information to support identification (e.g., size, colour, shape, surface appearance, etc.) as different species often produce distinctly different colony morphologies.

Selective and differential media can be used to isolate specific microorganisms or a group of microorganisms. Selective media promotes the growth of a particular bacterium of interest by inhibiting the growth of unwanted bacteria. 

1. Environmental Requirements: 

The environmental requirements for an organism to grow (such as oxygen concentration and temperature) can also aid presumptive identification; however, as with colonial morphology, this information alone cannot be relied upon to make a definitive identification.

1. Nutritional Requirements:

These are generally based on the nutritional requirements of the isolate and its ability to grow in the presence of certain inhibitors such as antibiotics or salts. 

The number and types of tests required to identify an isolate depend on several factors, including:

• The type of bacteria to be identified.  
• The significance of the isolate to the manufacturing process. 

Commercial test kits have been developed that incorporate miniaturised versions of the traditional individual biochemical tests and are available in both manual and automated formats.

CHEMOTAXONOMIC MICROBIAL IDENTIFICATION METHODS

The commonly used chemical characteristics for the identification and classification of bacteria include:

• Fatty acid profile 

Fatty acids are organic components of the phospholipid bilayer of the bacterial membrane, and their chemical and physical properties influence a wide range of biochemical functions. The unique combination of fatty acids found in different bacterial species makes fatty acid profiling a valuable tool for microbial identification.

A gas-liquid chromatography method has been developed to identify bacteria by fatty acid methyl ester (FAME) analysis.

• Protein profile

The protein profile of a bacterial strain reflects its genome; therefore, analysis of cellular protein content can contribute to bacterial identification and classification.   

GENOTYPIC MICROBIAL IDENTIFICATION METHODS

Genotypic methods are based on the analysis of an organism’s genetic material, such as DNA. They are generally regarded as the gold standard for microbial identification as they are considered more reliable than culture-dependent phenotypic methods.

However, genotypic methods are not always 100% accurate, partly due to imitations in the reference databases, and phenotypic data should therefore be used to support and confirm genotypic identifications.

Genotypic microbial identification includes:

• Nucleic Acid Base Sequencing. 
• Strain Typing.
• Repetitive Sequence-Based PCR. 
• Ribotyping.

HOW DO YOU CHOOSE WHAT SYSTEM TO USE?

When choosing which technology to use, the following factors are taken into consideration:

7 COMMON ISSUES WITH BACTERIAL IDENTIFICATION

1. No single identification system is 100% accurate all the time: All identification systems have limitations, and the user should be able to identify and defend the choice of the system used.
2. Over-reliance on the method: It is important to review all available data for the isolate being identified.
3. Over-identification: Avoid unnecessary identification- there must be a clear purpose for the data collected and an understanding of its significance.
4. Not doing the basics: Basic microscopic and macroscopic observations are essential for determining the appropriate next steps in identification and for supporting and confirming the results obtained from more advanced identification systems.
5. Inadequate database: Some identification systems cannot identify certain organisms; therefore, it’s important to ensure that the selected system has a sufficiently comprehensive and appropriate database for the types of organisms being recovered.   
6. Taxonomic changes and closely related species: Historically, new species have often been described based on data from a single strain. However, taxonomic classifications are subject to change and must be kept up to date. In addition, different species within the same genus may be very closely related and can be difficult to differentiate using a single identification system.  
7. Data integrity: In addition to other aspects of microbiology, bacterial identification methods and processes should be subject to a data integrity review.

CONCLUSION

Correct and reliable identification is essential for determining appropriate corrective and preventive actions following the recovery of an isolate from a product or facility. However, microbial identification can be challenging, and no identification system is 100% accurate.

Therefore, it is good practice to define a user requirements specification and select the system that is most appropriate for the needs of the laboratory.

This guide to bacterial identification was developed by Dr. Anna Lovatt and Dr. Tim Sandle following a request from Pharmig members at the Pharmig Annual Conference. 

FAQs

1. What is bacterial identification and why is it important in pharmaceutical manufacturing?

Bacterial identification is the process of determining the genus and species of a microorganism recovered from a product, raw material, or manufacturing environment. In pharmaceutical manufacturing, accurate microbial identification is essential for contamination investigation, root cause analysis, regulatory compliance, and implementation of corrective and preventive actions (CAPA). It helps assess product risk, patient safety impact, and environmental monitoring trends.

2. What are the main microbial identification methods used in microbiology laboratories?

Microbial identification methods are broadly categorized into:

• Phenotypic methods (based on morphology, biochemical reactions, and growth characteristics)
• Chemotaxonomic methods (based on fatty acid profiles and protein analysis)
• Genotypic methods (based on DNA sequencing and molecular analysis)

A polyphasic approach combining phenotypic and genotypic methods is often used to improve accuracy and reliability in bacterial identification.

3. How does genotypic microbial identification differ from phenotypic identification?

Phenotypic microbial identification relies on observable characteristics such as colony morphology, Gram staining, biochemical reactions, and environmental growth requirements.

Genotypic microbial identification, on the other hand, analyzes the organism’s genetic material through techniques such as nucleic acid sequencing, strain typing, ribotyping, and PCR-based methods.

Genotypic methods are generally considered the gold standard for microbial identification, but phenotypic data is still important to support and confirm results, especially when database limitations exist.

4. What factors should be considered when selecting a bacterial identification system?

When selecting a bacterial identification system, laboratories should evaluate:

• Purpose of testing and required turnaround time
• Database size and compatibility with existing systems
• Infrastructure and training requirements
• Capital and per-test costs
• Validation, regulatory compliance, and data integrity requirements

Defining a User Requirements Specification (URS) ensures that the chosen microbial identification system aligns with operational and quality needs.

5. What are the common challenges in bacterial identification?

Common challenges in bacterial identification include:

• No system being 100% accurate
• Over-reliance on a single identification method
• Inadequate or outdated reference databases
• Taxonomic changes and closely related species
• Data integrity concerns
• Skipping fundamental microscopic and colony morphology observations

To mitigate these issues, microbiology laboratories should apply a systematic, evidence-based, and polyphasic identification approach supported by proper documentation and quality controls.