ISOLATION OF PURE CULTURE ORGANISMS

• Pure culture


  Culture that contains only one species of bacteria
• Pure cultures


  • Required for subsequent procedures used to identify and characterize bacteria
  • Ability to select pure (individual) colonies is one of the first and most important steps required for bacterial identification and characterization
• Serial dilution


  1. Series of sequential dilutions

  2. Dilution is the process of decreasing the bacterial population to a certain concentration

  
• Tenfold serial dilution


  1. 10, 1:100, etc.
• How to make 1:10 dilution

  1 mL of your bacterial broth + 9mL of your sterile NSS
• How to get 1:100 dilution

  Get 1mL of your 1:10 dilution and transfer it into the next tube containing 9mL of sterile NSS
• How to get 1:1000 dilution

  Get 1mL of your 1:100 dilution and transfer it into the next tube containing 9mL of sterile NSS
• NSS = Normal Saline Solution
• Since 1:10 is the least diluted sample, you will have more bacterial growth as compared to the most diluted sample, meaning there is less concentration of your bacteria

  
• Pour plate


  Quantitative method
• Nutrient agar


  • A simple culture medium used for the isolation of non-fastidious organisms
  • Safe to use in the school laboratory because it doesn't selectively grow pathogenic bacteria

  
• Composition of nutrient agar

  • Pepton Digest of animal tissue: 5gm/L
  • Beef extract: 1.5 gm/L
  • NaCl: 5gm/L
  • Agar: 15 gm/L
  • Sterile water
• Procedure for pour plate

  1. Get bacterial suspension with a determined dilution

  2. Using a sterile pipette, get 1mL from the bacterial suspension and place inside the petri dish

  3. Get a tube of melted nutrient agar and allow to cool to the point that it can be held comfortable in the hand

  4. Remove the cotton plug from the melted nutrient agar tube and flame the mouth of the tube (aseptic technique prevents contamination from the environment). Lift the lid of the petri dish just enough to pour the agar into the dish

  5. Gently swirl the petri dish so that the agar will be evenly distributed. Allow the agar to hard

  6. Incubate at 37 degrees Celsius for 24-48 hours in order to give time for your bacteria to grow
• The next day you will see growth in your agar plate and you will now proceed to colony counting using a Quebec colony counter
• Calculating colony count


  1. Using a Quebec colony counter, count the colonies found on the plate

  2. Colony count x [Reciprocal of dilution] = CFU/mL (colony forming unit per mL)

  3. If the number of colonies is TNTC (Too numerous to count) use this formula instead: Average # of colonies in 5 squares x 62.5 x Reciprocal of dilution = CFU/mL
• Quebec colony counter


  • Used to quantitate colonies
  • Count the surface and subsurface colonies
• Four quadrant streak plate method

  1. Isolating bacteria into individual colonies to obtain pure colonies

  2. Inoculating loop or needle is streaked over an agar surface

  3. On the initial region of the streak, many microorganisms are deposited resulting in confluent growth or the growth of culture over the entire surface of the streaked area

  4. The loop is sterilized between streaking different sections, or zones and thus lesser microorganisms are deposited as the streaking progresses

  5. The streaking process will dilute out the sample that was placed in the initial region of the agar surface

  
• The 1st quadrant usually has the most growth, and then the succeeding quadrants will have lesser growth. In the 3rd quadrant, you could see individual colonies. So, these are pure culture colonies. The 4th quadrant contains your individual colonies. These are your pure colonies. This is the pure culture of your microorganism. This is the one that will be used for the identification of your microorganism since this is a pure culture.

  
• Procedure for four quadrant streak plate

  1. In the first quadrant, you will get three (3) loopful from your bacterial suspension and then you streaked in this manner. Turn 45 degrees and then streak

  2. Touch the first quadrant 2-3 times

  3. Before you proceed to your second quadrant make sure you are going to sterilize the inoculating loop

  4. Hit the inoculating loop in between quadrants in order for you to isolate the pure culture and in order for you to prevent contamination

  5. Streak first the first quadrant and then hit your inoculating loop

  6. Then touch your first quadrant 2-3 times and streak for your second quadrant and then turn it 45 degrees

  7. Streak again for your third quadrant. Touch 2-3 times only

  8. And then for your fourth quadrant, since this is the quadrant where you will find the pure culture or your individual colonies. You will touch your third quadrant only once

  
• Criteria to characterize bacterial growth

  • Colony size
  • Colony pigmentation
  • Colony shape
  • Colony surface appearance
  • Changes in agar media resulting from bacterial growth
  • Pattern on blood agar
  • Odor

  
• Bacterial colony


  • Size
  • Pigmentation
  • Shape
  • Surface appearance
  • Changes in agar media
  • Pattern on blood agar
  • Odor

  
• Streak plate technique


  1. Obtain inoculum

  2. Flame loop

  3. Streak in quadrants

  4. Incubate inverted

  5. Observe colonies

  
• Escherichia coli


  • Greenish metallic sheen on EMB agar
• Klebsiella pneumoniae


  • Large pink mucoid colonies on MacConkey agar
• Medtech can identify possible organisms by looking at colony growth
• Isolating bacteria


  1. Obtain sample by swabbing

  2. Suspend in sterile NSS

  3. Streak using four quadrant method

  4. Incubate plate upside down

  
• Incubating plate upside down

  Prevents moisture from lid dropping onto agar and causing separation of colonies
• Check for growth after 24-48 hours of incubation
• Pure culture


  • All colonies are identical
• Mixed culture


  • Colonies of different bacteria present
• Properly discard plates to be autoclaved after recording data