Culture: Identification and Antimicrobial Susceptibility Test

Explore culture in microbiology: a foundational technique used to grow and isolate bacteria, fungi, and other microbes under controlled conditions. Culturing supports pathogen identification, antibiotic sensitivity testing, and disease diagnosis critical for nursing, laboratory science, and infection control.

Introduction

Microbial culture and antimicrobial susceptibility testing are foundational practices in clinical microbiology, essential for the accurate diagnosis, effective treatment, and management of infectious diseases. The ability to isolate, identify, and test the susceptibility of microorganisms not only informs patient care but also supports epidemiological surveillance and antimicrobial stewardship. With the rise of antimicrobial resistance and emerging pathogens, these laboratory techniques have gained renewed significance in both routine diagnostics and public health interventions.

Culture Media

Culture media are nutrient-rich preparations designed to support the growth of microorganisms in laboratory settings. The selection of appropriate culture media is critical for successful isolation, identification, and further testing of pathogens. Culture media can be classified based on their physical state, nutritional composition, and specific applications.

Physical Types of Culture Media

• Liquid Media (Broth): These are fluid preparations such as nutrient broth and tryptic soy broth, used for the cultivation of bacteria in bulk and for sub-culturing purposes.
• Semi-solid Media: Contain lower concentrations of agar (usually 0.2–0.5%) and are used to detect bacterial motility and facilitate the growth of microaerophilic organisms.
• Solid Media: Typically contain 1.5–2% agar, providing a surface for the isolation of discrete colonies. Examples include nutrient agar, blood agar, and MacConkey agar.

Classification Based on Nutritional Content

• Simple (Basal) Media: Provide basic nutrients required for the growth of non-fastidious organisms. Examples: nutrient broth, nutrient agar.
• Complex Media: Contain additional nutrients such as peptones, yeast extract, and meat extract, supporting a wider range of bacteria. Examples: brain heart infusion broth, trypticase soy agar.
• Enriched Media: Basal media supplemented with blood, serum, or other growth factors for fastidious organisms. Example: blood agar, chocolate agar.
• Selective Media: Contain agents that inhibit certain microbes while permitting the growth of others. Examples: MacConkey agar (selects for Gram-negative bacteria), mannitol salt agar (selects for staphylococci).
• Differential Media: Incorporate indicators that reveal differences between organisms based on metabolic activities. Examples: MacConkey agar (lactose fermenters turn pink), XLD agar.
• Transport Media: Formulated to preserve viability of organisms during transport without promoting growth. Examples: Stuart’s and Amies transport media.

Composition of Common Culture Media

Blood Culture Media

Blood cultures are critical for the diagnosis of bacteraemia, septicaemia, and endocarditis. The detection of pathogens in blood requires specialised media and meticulous protocols to ensure sensitivity and specificity.

Types of Blood Culture Media

• Conventional Blood Culture Broths: Include brain heart infusion broth, tryptic soy broth, and glucose broth. These support a wide variety of bacteria and fungi.
• Enriched Broths: Contain supplements such as sodium polyanethol sulphonate (SPS) to neutralise antimicrobial agents in patient blood and support the growth of fastidious organisms.
• Anaerobic and Aerobic Media: Blood cultures are typically collected in pairs—one bottle containing media for aerobic organisms and another for anaerobes.
• Automated Blood Culture Systems: Commercially available bottles (e.g., BACTEC, BacT/ALERT) are pre-filled with enriched media and designed for continuous monitoring of microbial growth.

Protocols for Blood Culture Collection and Processing

1. Patient Preparation: Proper antisepsis is vital. The venepuncture site is cleaned with 70% alcohol followed by an iodine-based solution or chlorhexidine.
2. Sample Collection: Ideally, 8–10 ml of blood for adults and 1–3 ml for children is collected per bottle. Multiple sets (from different sites) are recommended to improve yield and distinguish contamination from true infection.
3. Inoculation and Incubation: Blood is immediately inoculated into culture bottles containing appropriate media. Bottles are incubated at 35–37°C, with regular monitoring for signs of growth (e.g., turbidity, gas production, automated detection of CO2).
4. Subculture and Identification: Positive cultures are subcultured onto solid media for isolation, followed by identification and susceptibility testing.

Prevention of Contamination

Strict aseptic technique, use of sterile equipment, and proper skin disinfection are essential to prevent contamination, which can lead to false-positive results and inappropriate management.

Culture Methods

The choice of culture method depends on the specimen type, suspected pathogen, and the purpose of isolation. Each technique has specific advantages for obtaining pure cultures, quantification, or selective enrichment.

Streak Plate Method

The streak plate technique is the most common method for isolating pure colonies from clinical specimens. Using a sterile loop, the sample is sequentially streaked over the surface of a solid agar plate, diluting the inoculum with each quadrant. This results in discrete, isolated colonies in the final sector, which can be further subcultured for identification.

Pour Plate Method

This technique involves mixing a diluted specimen with molten agar and pouring it into a Petri dish. As the agar solidifies, colonies develop both on the surface and within the medium. The pour plate is particularly useful for quantifying viable organisms in a sample, such as in urine cultures or environmental microbiology.

Spread Plate Method

A small, measured volume of diluted specimen is placed on the surface of a solid agar plate and evenly spread using a sterile spreader (e.g., glass rod or disposable plastic spreader). The spread plate is ideal for counting colony-forming units and for isolation when the microbial load is low.

Enrichment Culture

Enrichment techniques use specialised media or incubation conditions to favour the growth of specific organisms while suppressing others. For instance, selenite F broth enriches for Salmonella in stool samples, while alkaline peptone water is used for Vibrio cholerae. After incubation, subcultures are performed on selective solid media for further identification.

Culture Identification

Once pure colonies are obtained, accurate identification is essential for guiding therapy and understanding epidemiology. Identification combines classical morphological and biochemical methods with modern molecular techniques.

Morphological Identification

• Colony Morphology: Observations include size, shape, colour, elevation, edge, surface texture, and haemolysis on blood agar. Distinctive features may suggest specific genera (e.g., golden-yellow colonies of Staphylococcus aureus, swarming of Proteus species).
• Microscopic Examination: Gram staining is the primary step, differentiating bacteria into Gram-positive (purple) and Gram-negative (pink/red) based on cell wall structure. Other stains (e.g., Ziehl-Neelsen for acid-fast bacilli, India ink for Cryptococcus) may be used for specific organisms.

Biochemical Identification

• Carbohydrate Fermentation: Ability to ferment sugars (e.g., glucose, lactose, sucrose) is assessed by acid and gas production, often detected by colour change in pH indicators.
• Enzyme Production: Tests for catalase, oxidase, urease, coagulase, and others provide rapid differentiation among bacterial species.
• Utilisation of Substrates: Citrate utilisation, indole production, and hydrogen sulphide generation are key tests in differentiating enteric bacteria.
• Commercial Identification Kits: Systems such as API strips and VITEK cards offer panels of biochemical tests for rapid identification.

Molecular Identification

• Polymerase Chain Reaction (PCR): Amplifies organism-specific DNA or RNA sequences for precise identification, often within hours.
• Sequencing: 16S rRNA gene sequencing provides definitive identification, especially for rare or unusual isolates.
• MALDI-TOF Mass Spectrometry: Matrix-assisted laser desorption/ionisation time-of-flight analysis rapidly identifies bacteria and fungi based on unique protein profiles.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing (AST) determines the sensitivity or resistance of microbial isolates to specific antimicrobial agents. AST is crucial for:

• Guiding effective antibiotic therapy for individual patients
• Detecting emerging resistance patterns for infection control
• Supporting public health surveillance and antimicrobial stewardship

The results of AST inform clinicians about the most appropriate antimicrobial agents, helping to avoid unnecessary use, minimise toxicity, and reduce the development of resistance.

Methods of Antimicrobial Susceptibility Testing

Several laboratory methods are employed in clinical microbiology to assess the susceptibility of pathogens to antibiotics. The choice of method depends on the organism, clinical context, and available resources.

Disk Diffusion Method (Kirby-Bauer Technique)

This is the most widely used qualitative method for routine AST. The procedure involves:

1. Preparing a lawn culture of the test organism on Mueller-Hinton agar plate.
2. Placing standardised antibiotic-impregnated disks on the surface.
3. Incubating the plate at 35–37°C for 16–18 hours.
4. Measuring the diameter of zones of inhibition around each disk.
5. Interpreting results as sensitive, intermediate, or resistant based on standard charts (e.g., CLSI, EUCAST guidelines).

Advantages include simplicity, low cost, and the ability to test multiple antibiotics simultaneously. Limitations include lack of quantitative MIC data and potential variability with fastidious or slow-growing organisms.

Broth Dilution Methods

Broth dilution techniques provide quantitative measurement of the minimum inhibitory concentration (MIC)—the lowest concentration of antibiotic that inhibits visible growth.

• Macrobroth Dilution: Serial dilutions of antibiotics are prepared in test tubes with broth, inoculated with the test organism, and incubated. The MIC is the lowest concentration showing no turbidity.
• Microbroth Dilution: Similar to macrobroth, but performed in microtiter plates, allowing simultaneous testing of multiple antibiotics and isolates. This is the standard for reference laboratories and automated systems.

Agar Dilution Method

In this method, varying concentrations of antibiotic are incorporated into agar plates. The test organism is spot-inoculated onto each plate, and after incubation, the MIC is determined as the lowest antibiotic concentration preventing visible growth.

E-test (Epsilometer Test)

The E-test combines the principles of disk diffusion and dilution. A plastic strip impregnated with a gradient of antibiotic concentrations is placed on an inoculated agar plate. After incubation, an elliptical zone of inhibition forms, and the MIC is read where the zone intersects the scale on the strip.

The E-test is user-friendly and provides precise MIC values, but is more expensive than traditional disk diffusion.

Automated Systems

Automated instruments such as VITEK, Phoenix, and MicroScan offer rapid and standardised AST by integrating microdilution, growth detection, and result interpretation. These systems reduce manual errors, increase throughput, and often interface directly with laboratory information systems for seamless reporting.

Interpretation of Results

Interpretation of AST results requires careful consideration of laboratory data, clinical context, and established guidelines. Key points include:

• Breakpoints: Clinical breakpoints define the MIC or zone diameter thresholds for categorising organisms as susceptible, intermediate, or resistant, based on achievable drug concentrations in the body and clinical efficacy.
• Reporting: Only clinically relevant antibiotics should be reported, tailored to the infection site and patient factors. Reporting includes organism identity, susceptibility profile, and, if applicable, detection of specific resistance mechanisms (e.g., ESBL, MRSA, carbapenemase production).
• Quality Control: Use of standard control strains ensures accuracy and reliability of test results. Discrepancies should prompt repeat testing or alternative methods.
• Communication: Prompt and clear communication of critical results (e.g., detection of multidrug-resistant organisms) to clinicians is essential for timely intervention.

Challenges and Advances

Despite advances, several challenges persist in microbial culture and susceptibility testing:

• Time to Results: Traditional culture and AST methods may take 24–72 hours, delaying targeted therapy.
• Fastidious and Slow-Growing Organisms: Some pathogens are difficult to culture or require prolonged incubation (e.g., mycobacteria, certain fungi).
• Polymicrobial Infections: Mixed cultures can complicate identification and susceptibility interpretation.
• Emergence of Resistance: Continuous evolution of resistance mechanisms necessitates regular updates in testing protocols and interpretation guidelines.

Recent advancements aim to address these limitations:

• Rapid Molecular Diagnostics: PCR and nucleic acid amplification tests enable direct detection and identification of pathogens and resistance genes from clinical specimens, often within hours.
• Mass Spectrometry: MALDI-TOF has revolutionised identification by providing rapid, accurate results for a wide range of bacteria and fungi.
• Next-Generation Sequencing (NGS): Offers comprehensive pathogen profiling and detection of resistance determinants but is currently limited by cost and complexity.
• Point-of-Care Testing: Portable devices and microfluidic platforms are being developed for decentralised, rapid diagnostics, particularly in resource-limited settings.
• Artificial Intelligence and Automation: Machine learning algorithms are increasingly applied to interpret complex data and streamline laboratory workflows.

Conclusion

Microbial culture, identification, and antimicrobial susceptibility testing remain cornerstones of clinical microbiology, underpinning the diagnosis and management of infectious diseases. Mastery of culture media selection, blood culture protocols, culture methods, and identification techniques is essential for accurate pathogen detection. Antimicrobial susceptibility testing guides rational therapy, supports antimicrobial stewardship, and plays a pivotal role in combating the global threat of antibiotic resistance.

Ongoing technological innovations promise to enhance the speed, accuracy, and comprehensiveness of microbiological diagnostics. However, these advances must be balanced with rigorous quality control, thoughtful interpretation, and effective communication with clinical teams. By integrating traditional and emerging approaches, laboratory professionals and clinicians can ensure optimal patient outcomes and contribute to the broader effort of preserving antimicrobial efficacy for future generations.

REFERENCES

1. Apurba S Sastry, Essential Applied Microbiology for Nurses including Infection Control and Safety, First Edition 2022, Jaypee Publishers, ISBN: 978-9354659386
2. Joanne Willey, Prescott’s Microbiology, 11th Edition, 2019, Innox Publishers, ASIN- B0FM8CVYL4.
3. Anju Dhir, Textbook of Applied Microbiology including Infection Control and Safety, 2nd Edition, December 2022, CBS Publishers and Distributors, ISBN: 978-9390619450
4. Gerard J. Tortora, Microbiology: An Introduction 13th Edition, 2019, Published by Pearson, ISBN: 978-0134688640 
5. Durrant RJ, Doig AK, Buxton RL, Fenn JP. Microbiology Education in Nursing Practice. J Microbiol Biol Educ. 2017 Sep 1;18(2):18.2.43. https://pmc.ncbi.nlm.nih.gov/articles/PMC5577971/

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