Evaluation of Asceptic technique- by nutrient agar plate and nutrient broth

1. PRACTICAL CLASS-3

2. Preventing the entry of microorganisms into sterile
media/materials and cultures from the environment
and preventing the escape of microorganisms from the
culture into the environment

5. 1. Disinfect the work area before
starting to reduce potential
contaminants on the bench top, and
after work is finished to protect
others from possible contamination.

6. 2. Flame the inoculating loop before
and after making a transfer of
bacteria from one container to
another.Never lay an inoculating
loop on the bench top if you are not
sure it has been flamed first. When
in doubt, flame the loop again.

7. 3. Flame the opening of glass containers before
removing bacteria from them and again after
bacteria have been removed. Likewise, flame
the opening before transferring bacteria to a
container and again after the transfer is
completed.

8. 4. Do not lay the cap of containers of
bacteria on the bench top while
bacteria are removed from or
transferred to the container. The cap
should remain under your control
throughout the transfer.

9. 5. Work quickly and efficiently to minimize the time the
culture is exposed to the environment.

10. It is a must to avoid cross ventilation, running a
fan during asceptic operations
It is advisable to avoid talking, coughing,
sneezing during asceptic operations

12. Evaluation of asceptic technique of self using a
sterile Nutrient agar plate and an inoculation
loop
When a sterile agar medium is used for asceptic
transfer,, if any contamination takes place due to
improper technique, contaminants will grow in the
medium as colonies. If asceptic conditions are followed
no growth occurs

17. Ensure that the work bench is cleaned and disinfected.
Wash your hands with soap and water & apply
disinfectant to hand after wiping the hands
Collect the sterilized material
Label the petridish around the sides or on the bottom of
the plate
Sterilize the inoculation loop on the flame

18. Warm the nutrient agar plate on the flame
Lift the top plate of nutrient agar plate and draw the lines with
inoculation loop on the surface of agar plate
Close the lid of the nutrient agar plate

19. Sterilize the inoculation loop on the flame
Incubate nutrient agar plate at 30֯ C in an
incubator for 24 hours
Observe the growth in the form of colonies
Record the results as number of colonies
present or absent, if present see whether they
are on the lines or away from the lines

23. No growth in
the agar plate
was observed
Growth in the
agar plates was
observed

24. As no growth was
observed it indicates
that I have followed
asceptic conditions
properly
Growth was observed it
indicates that I have not
followed asceptic
conditions

26. Evaluation of asceptic technique of self
using a sterile Nutrient broth tubes and an
inoculation loop
When a sterile agar medium is used for asceptic
transfer,, if any contamination takes place due to
improper technique, contaminants will grow in the
medium as colonies. If asceptic conditions are followed
no growth occurs

28. Ensure that the work bench is cleaned and disinfected.
Wash your hands with soap and water & apply disinfectant
to hand after wiping the hands
Collect the sterilized material
Arrange the culture tube and sterile broth tube in
the clean test tube rack.
Label the material properly
Keep the rack on a disinfected
workbench on your left side

29. Sterilize the inoculation loop by heating the
nichrome wire to red hot on a Bunsen flame
Take the culture tube into your left hand and remove
the cotton plug/cap with right hand while holding the
sterilized inoculation loop near the flame
Heat the mouth of the culture tube by turning it on
flame with your left hand

30. Keep the culture tube in an angle to the work bench and
nearer to the flame
Insert the sterilized inoculation loop into culture
tube and pick up a loopful of culture
Once again heat the culture tube mouth on the flame
before keeping back the cotton plug/cap, while
holding the loop near the flame
Leave the culture tube on the rack and pick up the
sterile broth tube into which the culture is to be
inoculated

31. Remove the cotton plug/cap, heat the mouth of the broth tube, and
transfer the loopful of culture on the inoculation loop into broth
check whether the loop contains film of culture or not
Remove the inoculation loop, heat the mouth of the broth tube,
replace the cotton plug/cap and leave the broth tube on the rack
Sterilize the inoculation loop and leave it on the work bench.
Leave the inoculated broth tube in an incubator at 30֯ C and the
culture tube in a refrigerator if it is to be preserved further

32. Discard the waste material, if any, into proper waste
colletor
Clean the work bench and disinfect if necessary and
wash your hands with soap immediately after
completion of work

33. INCINERATE THE INOCULTION
LOOP
ORGANISM IN CULTURE ARE
DISPERSED BY SHAKING TUBE

34. TUBE CAP IS REMOVED AND MOUTH
OF THE TUBE IS FLAMED
A LOOPFUL OF ORGANISM IS
REMOVED FROM TUBE

35. LOOP IS REMOVED FROM CULTURE AND
TUBE MOUTH IS FLAMED
TUBE IS CLOSED WITH CAP

36. OPEN THE CAP OF OTHER TUBE AND SHOW IT
TO FLAME
INOCULATE THE CULTURE INTO THE
TUBE

37. INCINERATE THE INOCULATION
LOOP
AGAIN SHOW THE TUBE TO THE
FLAME AND CLOSE THE CAP

38. No growth in the
nutrient broth tubes
was observed
Growth in the
broth tubes was
observed

40. As no growth was
observed it indicates
that I have followed
the asceptic conditions
Growth was observed
in the tubes due to I
have not followed
asceptic conditions

42. 1) Why does an inoculation loop is heated initially in flame from the fixed end at an
angle?
2) Why do you sterilize an inoculation loop in a flame after asceptic transfer of a
culture?
3) Why should a sterile pipette be passed through the flame soon after its removal
from the sterile container?
4) Why should a sterile pipette be left in a tray containing disinfectant solution?

43. Experiment number and date
Title of the experiment
Theory part of experiment; for example : definition,
types, etc.,.
Aim
Principle
Materials required
Procedure: step wise
Flow chart along with diagrams
PATTERN FOR WRITING IN PRACTICAL
RECORD (A4 SHEETS)