Microorganism Cultivation and Isolation Techniques

Posted on Dec 13, 2024 in Biology

Nutritive Agar for Undemanding Microorganisms

Peptone Water

Liquid culture medium for prior non-selective isolation of bacteria. Incubation: Approximately 18 hours at 37ºC.

Blood Agar, 6.8 pH

Isolation, cultivation, and identification of various bacteria.

Potato Dextrose Agar

Cultivation, isolation, and determination of the number of yeast and mold organisms. Incubation: Up to 5 days at room temperature.

Sabouraud Agar

Clear culture medium recommended for the cultivation of dermatophytes and the sensitivity test of fungi.

SS Agar (Salmonella – Shigella)

Stimulates the growth of Salmonella and Shigella. Incubation: 18-24 hours at 37ºC.

MacConkey Agar

Isolation of Salmonella, Shigella, and coliform bacteria. Incubation: 18-24 hours at 37ºC.

XLD Agar (Xylose Lysine Deoxycholate)

Isolation and differentiation of pathogenic Enterobacteriaceae, Salmonella, and Shigella. Incubation: 48 hours at 37ºC.

Baird-Parker Agar

Isolation of Staphylococcus. Incubation: From 24-48 hours at 37ºC.

Cetrimide Agar

Culture for Pseudomonas. Incubation: Up to 48 hours at 42ºC.

Media Containing Sugars with pH Indicators

Study of fermentative properties of bacteria.

Kligler Medium

Identification of Enterobacteriaceae. Incubation: 48 hours at 37ºC.

Citrate Agar

Identification of microorganisms, Enterobacteriaceae. Incubation: 24-48 hours at 37ºC.

MR-VP Broth (Methyl Red-Voges Proskauer)

Biochemical differentiation, group Coli – Aerogenes. Incubation: up to 4 days at 37ºC.

Lactose Broth

Escherichia coli demonstrated by the formation of gas. Incubation: 24-48 hours at 37ºC.

Sample Processing by Nature

Solid Samples

a) Metacarpal or Metatarsal Marrow Seed: Saw the piece, burn the exposed bone surface with a spatula, and remove the sample with a pipette.

b) A Piece of Organ or Tissue: Extract a piece and grind it to reduce it to a paste.

c) Feces: Suspend in saline.

d) Hair and Scales for Identification of Fungi: Clear cut with lactophenol, as in plant scale samples.

e) Sporulated Anaerobic Tissue: Crush and suspend, heat at 80°C for 30-60 minutes to remove the contaminated flora.

Liquid Samples

a) Thick Abscesses: Suspend in saline.

b) Ear Drains: Suspend in saline.

c) Sputum: Decontamination with saline.

d) Urine: Dilute, centrifuge at 1500 rpm, and plant within two hours after collection and refrigeration.

e) Blood for Blood Culture: Use liquid anticoagulant.

f) Intestinal Contents: Centrifuge.

Enrichment

Physical Methods

a) Low Temperature of Incubation for Psychrophiles: 0° and 5°C, slowing the growth of other microorganisms.

b) High Temperature Incubation for Thermophiles: Above 55°C, preventing the growth of other microorganisms.

c) High Temperature for Spores: 80°C, destroys only spores.

Chemical Methods

a) Enrichment for Lactobacilli with Acid: MRS broth contains disodium phosphate, sodium acetate, ammonium citrate, and magnesium sulfate. The medium should have a final pH of 6.5. Isolate in the same medium, solidified cultures to a final pH of 5.5 for 18-24 hours at 35°-37°C.

b) Enrichment with Selenite for Salmonella: Sodium selenite at 0.4% concentration temporarily suppresses the growth of coliforms, while Salmonella growth continues. Plant directly on the medium incubated for 12-16 hours between 35° to 37°C. It should be insulated.

c) Enrichment with Sodium Chloride for Staphylococcus: NaCl enriched, 18-24 hours at 35°-37°C to isolate. Baird-Parker medium allows the detection, enumeration, and isolation. It contains lithium and potassium tellurite to remove other microorganisms. Incubate plates at 37°C for 24 hours.

d) Enrichment with Antibiotics: Penicillin, concentrations of 194 U/ml. Isolate.

e) Enrichment with Bile Salts for Intestinal Bacteria: Bile salts are added to MacConkey Agar or Salmonella-Shigella agar, and for Enterococcus on bile esculin agar.

Isolation

Isolation is the process of separating one or more microorganisms in a given sample to obtain pure cultures. For this purpose, it should be planted in solid nutrient media in Petri dishes in a way that ensures the growth of separated bacterial colonies and avoids mass development. In most cases, a colony presumably developed from a single cell is a sufficient guarantee of purity.

After obtaining a pure strain, it is possible to study its morphological and biochemical aspects to achieve its identification.

The isolation of microorganisms can be achieved by the following methods:

Mechanical Methods

Clock Method (Exhaustion Streaks): Distribute the inoculum in a small area near the border, drag a bit of bacteria to another area of the plate, and streak in a zig-zag pattern.

Dilution and Uniform Seeding: For very concentrated samples, make dilutions and sow evenly until separate colonies are obtained.

Monitoring of Crops

Label with date and number. Incubate at optimum temperature, with inverted Petri dishes to avoid dehydration. Observe at 24 hours.

Material Use

  1. Test tubes containing culture media and/or crops.
  2. Blood hemolysis tubes.
  3. Centrifuge tubes for liquid samples.
  4. Petri dishes for solid plating surface.
  5. Graduated pipettes for transferring exact volumes of liquids.
  6. Pasteur pipettes for transferring small and indeterminate volumes of liquids.
  7. Drigalski spatula for homogeneous seeding in Petri dishes.
  8. Erlenmeyer flasks for culture media preparation.
  9. Monod flasks for turbidimetric determination of bacterial growth.
  10. Kitasato flasks for sterilization by filtration of thermolabile substances.
  11. Cylinders for accurate measurement of volumes.
  12. Microscope slides for observations.
  13. Coverslips for microscopic observations.
  14. Excavated microscope slides for hanging drop observations.
  15. Durham bells for gas detection in liquid microbial cultures.
  16. Beakers for content and to prepare solutions.
  17. Loop handle for streak plating on solid medium and growing in liquid media.
  18. Straight loop for seeding by puncture.
  19. Millipore filter for sterilization of thermolabile liquids by filtration.
  20. Disk for recovery of antibiotics (antibiogram).
  21. Bunsen burner for sterilization and flaming red. Provides an aseptic environment for the workspace.
  22. Pipette holder (pipettor) to contain sterile pipettes.
  23. Basket to contain metallic material to be sterilized in an autoclave.
  24. Pipette disposal container for dirty and/or contaminated pipettes.