FOOD MICROBIOLOGY AND SAFETY (FDT 603)
LAB 3:
ASEPTIC LABORATORY PRACTICES
RISK OF CONTAMINATION
1. Take 5 plates of nutrient agar (NA) agar growth medium and label them 1-5.
Make sure the labels are on the bottom of the Petri dish. Lay these plates out on
the bench with the lids uppermost.
2. Take of the lids from the first 3 plates and lay them down on the bench. Replace
each of the lids in turn, at 20 seconds interval. Replace the first lid after 20
seconds, the second after 40 seconds and the final lid after 60 seconds.
3. For plate 4- Use one hand to lift the lid into the air and place the fingertips of
your other hand firmly on the surface of the medium in the plate without
breaking the media surface and replace the lid of plate 4.
4. For plate 5 - Wash your hands thoroughly with soap and water or detergent and
repeat the procedure you carried out for plate 4.
ASEPTIC USE OF PRE-STERILISED IMPLEMENTS
1. Take one fresh NA plates, divide each into half by drawing a line across a
diameter on the base. Now label one plate ‘PIPETTE’
2. Take one pipette and leave it on the bench surface. Remove a second pipette
and use it immediately. Only touch the pipette at the furthest end from the tip.
Carefully place the pipette pump at the end of the pipette and pass the tip of the
pipette three times quickly through the hot part of the Bunsen flame.
3. Now use the pipette to draw 0.5ml of distilled water from the fresh bottle and
expel 0.1 ml of this onto the surface of the medium in one half of the NA plate
labeled ‘PIPETTE’.
4. Take the second pipette from the bench. Place pipette filler without passing it
through the flame take a further 0.1ml of distilled water from the same bottle
and expel it on the surface of the medium in the second half of the same plate.
5. Allow this plate to rest undisturbed on the bench until the water has been
absorbed into the medium.
THE USE OF FLAME STERILIZED IMPLEMENTS
1. Take one fresh NA plate and mark the bottom of the plate with the word ‘LOOP’
and then divide the plate into 4 quarters. Label the sectors a-d.
2. Take a wire loop and press it gently to the surface of the medium in sector a.
Glide the loop backwards and forwards across the medium surface in this sector
only.
3. Hold the loop in the flame of a Bunsen burner at an angle of 20o to the flame, so
that a significant length of the wire is in the hottest part of the flame. Make sure
your fingers are well clear of the flame. Hold the wire there until it glows
white/yellow hot then remove it. After it stops glowing (5 seconds) touch it on
the surface of sector b in the same NA plate and glide it gently across the surface
of that sector as before.
4. Repeat the flame sterilization of the loop but this time after 5 seconds of cooling,
dip it gently in the bacterial suspension provided and again glide it across the
surface of the medium in the same plate in the sector c.
5. Leave sector d untouched as a control sector.
THE USE OF ALCOHOL STERILIZED IMPLEMENTS
Note: Do not place the jar of ethanol near the Bunsen flame or bring the flaming
spreader or scalpel back from the Bunsen flame towards the jar of ethanol.
1. Take a fresh NA plate and label its base ‘SPREADER’ then divide it into half by
drawing a single line across a diameter. Then take the glass spreader provided
and gently rub it over the surface of the medium in one half of the plate only.
2. Dip the spreader into the jar of 75% ethanol and leave it there for 30 seconds.
Remove the spreader, letting the excess ethanol drain back into the jar from the
free end of the spreader bar. Touch the spreader bar to the edge of the Bunsen
flame and move away from the flame immediately. The remaining alcohol will
ignite but will soon burn out. As soon as the flame disappears, touch the
spreader bar to the other half of the medium surface in the same plate and rub
the bar gently across the surface in that half only.
Note: The sterilization in this procedure is due to the antimicrobial effects of the
aqueous ethanol (70-90%) and NOT due to the heat from the flaming. The flame
merely removes the final traces of toxic ethanol prior to use of the spreader. The
tool must not be heated in he Bunsen flame, water must be present for the
sterilization effect of the ethanol to be maximal, so pure ethanol (100%) is not
used and solution below 70% are too week to be very effective.
3. Take a fresh NA plate and label its base as ‘SCALPEL’ divide it into two across a
diameter as before. Use a scalpel to cut a block of agar about 1 cm square from
the surface of the first half of the plate.
4. Sterilise the scalpel by dipping its blade into the 75% ethanol for about
30seconds, allowing the excess ethanol to drain back from the blade as you take
it out. Touch the top of the blade to the edge of the Bunsen flame and move the
scalpel away from the flame immediately. The excess ethanol will burn off
quickly and as soon as the flame disappears, use the scalpel to repeat the block
cutting exercise on the second half of the plate.
All the plates will be incubated at 370C till observation.