Culture Techniques

Transfer to Broth

1. Flame your loop

2. Dislodge bacterial culture broth via rolling between your palms

3. Perform the transfer

   • remove lid without placing on bench

   • flame tube rim

4. Replace the lids back on the tubes

   • first re-flame the tube rim

Transfer to Slant

1. Flame your loop

2. Dislodge bacterial culture broth via rolling between your palms

3. remove the cap and flame the tube

4. form and “S” shape from bottom to top along the media with inoculated loop

5. flame the tube and replace the cap

Transfer to Deep

1. Flame your transfer needle

2. Dislodge bacterial culture broth via rolling between your palms

3. remove the deep cap and flame the tube

4. inoculate the needle and then plunge it down the middle of the agar deep

5. flame the tube and replace the cap

Aseptic Bacterial Collection

1. flame your loop

2. collect bacteria from the broth

   • aseptically (flame after the lid comes off and before it goes back on)

3. collect bacteria from the plate

   • remove plate lid and dab sterile loop into bacteria, then replace the lid and invert the dish

4 Sector Streak Plate Inoculation

• tilt the media to face yourself

• avoid stabbing or gouging the agar

• flame your loop

• inoculate your loop and drag it over the 1st and 2nd sectors in one fluid motion

• flame your loop

• drag your sterile loop through the 2nd sector and streak it into the 3rd sector

• flame your loop

should leave you with 4 streaks

Lawn Inoculum

1. spread inoculated swab over the entire surface of the agar plate

   • back and forth movement

2. rotate the plate 60^o and swab again

   • same movement

3. rotate the plate another 60^o and swab for the last time

4. then swipe the swab around the perimeter of the plate

Detecting Coliforms: Eosin Methylene Blue (MB) Agar Plates

EMB plates are used after presumptive testing at the highest dilution

helps view coliform colonies - allows us to count the colonies

Detecting Coliforms: Membrane Filtration Technique

1. set up filtration equipment

   • attach filter trap to the vacuum source

   • place filter holder base on the filtering flask

   • attach filter flask to the trap

   • using sterile forceps place the filter on the filter holder

   • set the funnel on the filter holder and fasten it in place

2. filter the sample

   • shake the water sample to re-suspend all material

   • pour a measured volume into the funnel

   • turn on the vacuum and allow sample to pass through the filter

   • turn the vacuum off

3. inoculate the fliter

   • carefully remove the filter from its holder using sterile forceps

   • place the filter on EMB agar plate as it was placed on the holder

4. invert the plate and incubate

5. examine and count coliforms