Methods of Inoculation and Isolation Techniques

Methods of Inoculation and Isolation of Pure Culture

• The purpose of the experiment is to isolate single colonies of bacterial species from a mixed culture.
• Colonies are visible on the surface of solid media.
• A colony originates from a single microorganism.
• Obtaining single colonies ensures a pure culture.
• Methods include:
  • 4-quadrant streak method (4QSM).
  • T streak method (TS).
• 4QSM involves sequential dilutions of the sample over quadrants.
• The number of organisms decreases as the sample is streaked, resulting in isolated colonies in subsequent quadrants.
• Single isolated colonies generally appear in the third or fourth quadrant.

Spread Plate Method

• A microbiology lab technique isolates and counts viable microorganisms.
• A known volume of liquid sample is spread over solidified media.
• This method is used for isolating and counting culturable bacteria and fungi.

Pour Plate Method

• A technique for plating higher volumes of sample.
• Liquid inoculum is pipetted into empty petri dishes.
• Molten medium is poured over the inoculum.
• Colonies grow on the surface and within the medium.
• This method is ideal for samples with lower bacterial numbers.
• The difference between pour and spread plates: The molten agar is poured into the inoculum in the pour plate method and the inoculum is spread on the surface of the solidified agar in the spread plate method.

Agar Dilution Method

• A method to determine the minimum inhibitory concentration (MIC) of an antibiotic on bacteria.
• Different concentrations of a drug are prepared by diluting it with water.
• Media containing these drug dilutions are poured into agar plates.
• Plates are inoculated with bacteria.
• After incubation, observed expansion of fungi.

Broth Dilution Method

• This method determines antibiotic sensitivity.
• Containers holding identical volumes of broth with antimicrobial solution are inoculated with a known number of bacteria.
• Different concentrations of antimicrobial agents are present in the separate containers and are measured.
• Qualitative techniques are used- Kirby Bauer method (disc diffusion method), and incubated overnight.

Action of Dyes and Antibiotics

• Dyes are used in microbiology for their inhibitory and differential properties.
• Ex: carbol fuchsin differentiates between acid-fast and non-acid-fast bacteria.

Bacterial Resistance to Antibiotics

• Some bacterial species have developed resistance to antibiotics through mechanisms like enzyme release.
• Beta-lactamase is an example that breaks open the beta-lactam ring in penicillin.
• This inactivates the antibiotics, rendering them ineffective.

Procedure for Antibiotic Susceptibility Testing (Kirby-Bauer Method)

• Bacterial suspension is standardized with a swab, rotated inside a tube.
• Mueller Hinton agar (MHA) (pH 7.2-7.4) is prepared with a specific depth (4mm).
• The swab is streaked three times over the agar's surface in different directions.
• Sterile forceps place antimicrobial disks on the agar's surface.
• Press disks firmly for contact.
• Do not move disks after contact.
• Plates are inverted and incubated at 35°C-37°C for 16-18 hours.

McFarland 0.5 Standard

• Used to compare turbidity of bacterial suspension.
• Required for accurate antibiotic susceptibility testing.

Results and Interpretation

• Antibiotic susceptibility testing results are based on zone sizes:
  • Sensitive (S): Treatable with a normal dosage.
  • Intermediate (I): May respond with a higher dosage.
  • Resistant (R): The antibiotic is unlikely to be effective.
• Bactericidal drugs cause irreversible damage to bacteria, leading to death.
• Bacteriostatic drugs only prevent reproduction but don't kill the organism.