Unit 7: Culturing bacteria

Culturing Bacteria

• A culture is a group of microorganisms that grow in a medium.
• A medium is a solid, liquid, or semi-solid that supports the growth of microorganisms.
• Common media include broth, agar, and enriched media.
• Microbiologists can identify microorganisms by studying their characteristics and culturing them in appropriate media.

Culturing Bacteria

• Pure cultures contain cells from a single species of an organism.
• The chosen medium must meet the organism's enzyme and nutritional requirements.
• Streak plating is used to isolate pure samples.
• Pour plating is used to count bacteria and obtain pure cultures.
• Serial dilution is a technique involved in the pour plate method.
• Serial dilution helps isolate single colonies for further study.

Estimating Bacterial Numbers

• Choose a plate with between 30 and 300 colonies for accurate counting.
• Multiply the number of colonies by the reciprocal of the dilution factor to estimate total bacteria.
• Express the result in scientific notation.

Culture Media

• Culture media contains all nutrients needed for bacterial growth.
• Microbiologists must understand the nutritional needs of bacteria to provide them with the necessary nutrients in an artificial environment.
• No single media supports the growth of all bacteria.
• Anaerobic conditions may be needed for certain species.

Common Human Pathogenic Enterobacteriaceae

• This table lists several common human pathogenic Enterobacteriaceae species and their associated genus.

Differential Media

• Differential media allow for the differentiation of bacteria based on their characteristics.
• Eosin Methylene Blue (EMB) Agar:
  • Detects fecal coliform bacteria.
  • Contains Eosin Y and Methylene blue as pH indicators.
  • Inhibits Gram-positive bacteria.
  • Fermentable carbohydrates include lactose and sucrose.
  • Rapid fermenters produce green metallic colonies.
  • Medium fermenters produce pink colonies.
  • Non-coliforms produce colorless colonies.
• MacConkey Agar:
  • Selective and differential.
  • Selects for Gram-negative bacteria.
  • Contains lactose, crystal violet, bile salts, and neutral red indicator.
  • Lactose fermenters produce red colonies.
  • Non-lactose fermenters produce colorless colonies.
• Hektoen Enteric Agar:
  • Selective and differential.
  • Selects and differentiates Salmonella and Shigella from other Enterobacteriaceae.
  • Contains bile salts and bromothymol blue, inhibiting Gram-positive bacteria.
  • Ferrous compounds are present to indicate hydrogen sulfide gas production.
  • Salmonella colonies produce transparent green colonies with black centers.
  • Shigella colonies produce transparent green colonies.
  • Lactose fermenters produce salmon-colored colonies.
• Mannitol Salt Agar:
  • Selective and differential.
  • Isolates Staphylococci (Gram-positive bacteria).
  • Contains a high salt concentration (7.5%), inhibiting halophobic growth.
  • Pathogenic Staphylococci ferment mannitol, producing yellow colonies.
  • Non-pathogenic Staphylococci do not ferment mannitol, producing pink colonies.

Selective Media

• Bismuth Sulfite Agar:
  • Isolates Salmonella typhi (typhoid fever).
  • Contains bismuth sulfite and Brilliant green indicators, inhibiting Gram-positive and coliform bacteria.
  • The presence of S. typhi reduces sulfide, resulting in black or green metallic colonies.

Enrichment Media

• Tryptic Soy Agar and Broth (TSA/B):
  • General purpose non-selective media.
  • Provides ample nutrients for a wide range of bacterial species.
  • Frequently used in general microbiology labs.
• Blood Agar:
  • Contains general nutrients and 5% bovine (cow or sheep) blood.
  • Differentiates between hemolytic and non-hemolytic bacteria.
    • Alpha hemolytic bacteria: partially break down RBCs and produce a green color (e.g., Streptococcus pneumoniae).
    • Beta hemolytic bacteria: completely break down RBCs and cause clearing (e.g., Staphylococcus sp. and Streptococcus sp.).
    • Gamma hemolytic bacteria: can grow but do not break down RBCs (e.g., Staphylococcus sp.).
• Chocolate Agar:
  • Blood is added to tryptic soy agar and heated, causing the blood to lyse and release the nutrients.

Biochemical Tests

• Carbohydrate Fermentation Tests:
  • Use phenol red broth with a Durham tube.
  • Detects the fermentation of specific carbohydrates.
  • Positive result: broth turns yellow (acidic).
  • Negative result: broth stays red/pink (neutral).
  • Gas production is indicated by bubbles in the Durham tube.
• Litmus Test:
  • Uses litmus milk medium with litmus indicator.
  • Evaluates a bacteria's ability to use different milk products.
  • Color changes, gas production, or curdling of the media indicate positive results.
• Indole Production:
  • Uses tryptophan broth and Kovacs reagent.
  • Detects the ability of bacteria to break down tryptophan into indole.
  • Positive result: pink color forms on the surface of the broth.
• Methyl Red Test:
  • Uses Methyl Red-Voges-Proskauer (MRVP) media and Methyl Red solution.
  • Detects the ability of bacteria to produce and maintain acidic products from glucose fermentation.
  • Positive result: red color remains after adding the Methyl Red solution.
• Urease Test:
  • Uses media containing urea.
  • Detects the enzyme urease.
  • Positive result: ammonia production turns the litmus indicator basic (color change).
• Nitrate Reduction:
  • Uses media containing nitrate.
  • Detects the enzyme nitrate reductase.
  • Positive result: the production of nitrites is observed.
• Oxidase Reaction:
  • Uses TSA/B and oxidase reagent.
  • Detects the presence of cytochrome-oxidase, a key enzyme in the electron transport chain (ETC).
  • Positive result: a blue color appears.
  • This test helps determine a bacteria's ability to produce energy aerobically using the ETC.
• Egg-Yolk Reaction:
  • Uses egg-yolk agar.
  • Detects the enzyme lecithinase, which destroys animal tissue.
  • Positive result: a white precipitate forms around the colonies (indicates lecithinase activity).

Recall - Pour Plate Method

• A serial dilution is performed from a liquid specimen.
• Each dilution is used to pour a plate inoculated with bacteria in agar.
• Colonies are counted after incubation.
• A plate with single, pure colonies is selected for further growth.