Aseptic handling and transfer of microorganisms

Introduction
Aseptic handling is basic practices and procedures to prevent entry of unwanted organisms
while restricting the organism within specified material only. The microorganisms are routinely
transferred for cultivation, growth, and for large scale production of cell mass or metabolites. Aseptic
conditions are also maintained within surgical procedures, areas to prevent risk of infection. Such
operations are carried out in a controlled environment applying the strictest rules to restrict
unwarranted spread of microorganisms.
Microorganisms are carefully handled during varieties of procedures maintaining proper
aseptic conditions. Undesired entry of microorganism from air, media, working surface, utensil,
handling material, equipment, and human contact would contaminate the culture under study. Proper
aseptic handling technique can greatly minimize or even eliminate the risk of contamination and
escape of pathogenic microbes in the surrounding, so laboratory skill for handling microorganism is
this is important.
Rules
Faulty aseptic procedure can cause (1) Risk of contamination (2) Risk of Infection (3) Fluctuation
in result (4) Reduce speed and economy. Hence, the following rules are strictly implemented in
laboratory while handling live microbes.
1. Keep the working area clean by disinfection practice before and after transferring culture.
2. Sterilize the handling gadgets carefully, e.g. wire loops by flaming.
3. Carry out all the transfer within closely contained areas like Laminar air-flow system (horizontal
flow of filtered air), or one created between a pair of Bunsen burner (positive pressure from
combusted gas around flame) placed 6-8 inches apart.
4. Accomplish the transfer keeping the culture vessels open or exposed for minimum time duration by
being quick and efficient during transfer.
5. Restrict movements and air currents around the work space
6. Follow instructions carefully, and report and spillage and accidents instantly.
7. Periodic monitoring and disinfection of laboratory area and appliances is carried out.
Material
1. Handles with Nichrome wires (Straight and Loop), Sterile gloves, Laboratory coat
2. Disinfectant solution, Alcohol, Cotton swab
3. Microbial culture
4. Sterile distilled water 1 mL
5. Sterile Nutrient broth 50 mL
6. Nutrient Agar Plate
7. Nutrient Agar Slant
8. Sterile pipette (1.0 mL)
9. Discard Tray (with disinfecting solution)
Methods
(A) Prepare for Transfer
1. Disinfect table by wipe cleaning work surface with disinfectant
2. Wash hands with soap and water, recommended to wear sterile surgical gloves
3. Light two Bunsen burners 6-8” apart and adjust flames to fully oxidized state
4. Label all the material for transfer properly and set it nearby
5. Arrange discard tray in the nearby place
6. Heat the nichrome wire holding the handle between the thumb and first two fingers of right hand
and moving wire over flame from inner end towards distal end, finally hold it vertically over the
flame until glow red hot.
7. Hold the wire in sterile area between two flames for cooling. Do not take away until transfer is
accomplished.
(B) Make Culture Suspension
1. Pick source culture tube, hold the cotton plug from the last two fingers and palm of the left hand.
2. Keeping mouth of the tube between the flames, open the plug by rotating the tube and heat the
open mouth of the tube by rapidly passing over the flame.
3. Insert the sterilized wire-loop in to the tube and gently touch the medium to pick-up little cell mass.
4. Immediately flame the opening of the tube, insert back the cotton plug and place the tube in the
discard tray on one side.
5. Holding the nichrome wire loaded with the inoculum within the flame, pick target distilled water
tube and open its plug similarly in between the flames, pass the opening rapidly over the flame.
6. Insert loaded nichrome wire and homogenously suspend the cell-mass by shaking the loop gently in
to the distilled water. Flame the mouth of the tube, replace the cotton plug and place the tube in the
adjacent rack on another side.
7. Re-sterilize the wire-loop as done earlier.
(C) Inoculate Fresh Medium
Inoculate Agar Slant
1. Sterilize the straight wire as shown in steps (A) 6-7
2. Pick up inoculum as in Steps (B) 1-4 from the source culture suspension
3. Holding the nichrome wire loaded with the inoculum within the flame, pick target Nutrient agar
slant tube and open its plug in between the flames, pass the opening rapidly over the flame.
4. Stab the loaded nichrome wire gently straight in to the Nutrient agar slant until bottom. Flame the
mouth of the tube, replace cotton plug and place the tube in the adjacent rack on another side.
5. Re-sterilize the wire-loop as done earlier.
6. Repeat the same inoculation process another time using nichrome wire-loop and spreading culture
over surface of the agar slant
Inoculate Agar Plate
1. Sterilize the straight wire as shown in steps (A) 6-7
2. Pick up culture as in Steps (B) 1-4 from the suspension or cell-mass (from slant / plate)
3. Holding the nichrome wire loaded with the inoculum within the flame, pick target Nutrient agar
plate and hold it within the flames using 3rd and 4th fingers of the left hand.
4. Open the lid from one side holding it by thumb and 1st finger
5. Streak zig-zag lines with the loaded nichrome wire gently over the surface of Nutrient agar plate.
6. Close the lid and place the plate in the adjacent side.
7. Re-sterilize the wire-loop as done earlier.
Inoculate Nutrient Broth Flask
1. Take sterile pipette and open it keeping tip between flames
2. Open the source suspension as in Steps (B) 1-2
3. Insert tip of the pipette in the suspension and aspirate 0.3 mL of the suspension, flame and close
down the culture tube.
4. Holding the pipette loaded with the inoculum within the flame, pick target Nutrient broth flask and
open its plug in between the flames, pass the opening rapidly over the flame.
5. Empty the pipette in to the flask, flame the mouth of the flask, replace the cotton plug and place it
on another side.
6. Discard used pipette in the discard tray containing disinfecting solution.
(D) After Transfer
1. Disinfect table by wipe cleaning work surface with disinfectant
2. Place all inoculated material at proper place (e.g., incubation) labelling with user, content, date, etc.
3. Discard used material carefully as per instruction.