Group Number: 6 Date of Submission: September 10, 2022 Score

Contributors: Pahigon, Wina

Mara T.
Quijano, Xymoun Luis
Quioc, Patricia Angela
Razalan, Ronan P.
Reyes, Philip James
Ricio, Ashley Nicolle L.
Sabado, Jazlenne
San Diego, Trisha Joy
Class Code: 1115L Remarks:

ACTIVITY 4
INOCULATION AND INCUBATION TECHNIQUES

I. OBJECTIVES:

1. Apply the various methods of inoculating solid or liquid culture media.

2. Acquire the skills in aseptically transferring inoculums from cultures into media.
3. Know the various methods to maintain the required environmental conditions duringincubation.
4. Demonstrate the proper incubation techniques of bacterial cultures.

II. MATERIALS:

  Prepared agar plates – nutrient agar, BAM, TSA

  TSI slants; nutrient broth or TSB
  Inoculating loop and needle
  Bunsen burner
  Bacterial specimens/ stock cultures
  Incubator

III. PROCEDURE:

A. INOCULATING AN AGAR PLATE:

1. Heat an inoculating loop until red-hot. Cool for about 15 seconds and fish out a loopfulof the
specimen or culture.

2. Using your left hand, hold the Petri dish and partially open the lid of the plate.

3. Bring the inoculum in contact with the surface of the agar on the side away from you and streak
the surface back and forth from edge to edge in parallel lines moving towards you. Make the
streak lines close to each other and inoculate gently to preventdigging into the agar.

4. When the entire surface of the agar medium has been inoculated/streaked, cover the agar plate
and flame sterilize the needle. Pass the mouth of the tube over the flame before placing the
cap/cotton plug.

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 Fig. 1. Flaming an inoculating loop

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 Fig. 2. Flaming the neck of tubes

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 Fig. 3. Transferring microorganisms from broth culture

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 Fig. 4. Streak dilution method

Fig. 5. Pour plate method

Fig. 6. Spread plate method

B. INOCULATING THE AGAR SLANT:

1. Flame-sterilize an inoculating needle and obtain a small colony of culture from a solid medium
(plate or slant).

2. Using your left hand, hold the tube containing the agar slant and hold the cotton plug/screw cap
between the small and ring fingers of the right hand. NEVER LAY THE COVER OR COTTON PLUG ON
THE TABLE!

3. Pass the mouth of the tube through the Bunsen flame and inoculate the slant by first stabbing the
deep (or butt) of the tube at a depth of 2-3 mm. Withdraw the needle

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 and streak over the agar surface or slant with a back-and-forth “S” motion, starting
from the bottom going up. Take care not to gouge or injure the agar.

4. Remove the inoculating loop/needle then flame sterilize it and at the same time, passthe mouth of
the tube on the Bunsen flame.

5. Replace the cover of the tube and label appropriately.

Fig. 7. Slant to slant transfer using stab and streak method

C. INOCULATING A LIQUID OR BROTH MEDIUM:

1. Flame-sterilize an inoculating loop and fish out a loopful of the specimen. Open the broth tube and
gently tip to approximately 30-degree angle. Flame-sterilize the mouth of the tube and put in the
inoculating loop.

2. Dip the inoculating loop just above the point where the surface of the medium makes an acute
angle with the glass tube and rub the inner sides of the tube.

3. Return the culture media to an upright position to submerge the area of inoculation beneath the
surface of the medium.

4. Flame-sterilize the mouth of the tube and recap, at the same time, flame-sterilize the inoculating
loop.

Fig. 8. Broth to broth transfer

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D. INCUBATING THE BACTERIAL CULTURES:

1. Aerobic Technique:

a. As soon as the agar plates have been inoculated and labeled, invert the plates so that the
cover is at the bottom and the agar plate is on top. Place inside an incubator set at 37°C.

b. For the tube cultures, agar slants and broth tubes, they are placed on a tube rack or beaker
and also kept in an incubator.

c. All bacterial cultures are incubated at 37°C for 24 to 48 hours or until the next laboratory
period.

2. Capnophilic Technique:

a. Invert the plates and place the tubes on a test tube rack or in a beaker.

b. Place them inside the candle jar.

c. Apply petroleum jelly at the upper rim of the jar and its cover.

d. Light a candle and place it inside the candle jar. The petroleum jelly will serve as a seal
between the cover and the jar.

e. The lighted candle will continue to burn until the carbon dioxide concentration increases
enough to stop combustion.

f. Place the candle jar inside the incubator.

3. Anaerobic Technique:

a. Invert the plates and place the tubes on a test tube rack or in a beaker.

b. Place them into the GasPak jar.

c. Take one GasPak single-use Anaerobic Indicator, peel apart the foil at one end andpull halfway
down the envelope. The indicator will turn blue on exposure to air.

d. Place the indicator in the jar in such a manner that the wick will be visible outside the jar. The
reaction from blue to colorless, indicating anaerobic condition, may require several hours.

e. Cut the corner off of one GasPak Hydrogen-Carbon Dioxide Generator Envelope and place
on the jar in an upright position. Pipette 10 mL of water into the open corner of the envelope.

f. Place the GasPak lid on the jar, apply the clamp and secure it by turning the screw until it is
hand-tight.

g. Place the GasPak system inside the incubator

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 V. QUESTIONS:

1. Give the rationale of the following practices:

a. Flaming the mouth or lid of culture tubes or plates after opening and before
closingthem.

- Lids of culture tubes or plates are needed to be flamed after opening them to
promote a convection current that blows air away from their opening. This current
will remove and prevent microorganisms from entering the glassware, ensuring
that contamination does not occur as you do the inoculation process. However,
since the convection current is not permanent, reflaming these openings are done
again before closing to ensure that airborne contaminants that may have entered
during the process will also be taken out.

b. Flaming the inoculating needle or loop before and after inoculation.

- When attempting to pick up microbes, loops and needles must be sterilized in a

Bunsen burner flame until red hot and then cooled. This is done before any
inoculation to ensure any contaminating organisms are killed. As soon as you
place the red-hot loop into a culture transferred from broth, it sizzles. Wait one or
two seconds before withdrawing the inoculum loop from the tube because it will
naturally cool when it contacts the broth culture. The tip of the loop should get
hotter during burning. This is because it will contain culture after usage, which may
sputter on rapid heating and release small particles of culture, generating an
aerosol. Before and after the process, burn the inoculating needle or loop to kill
any microorganisms.

c. Holding caps, lids, or cotton plugs rather than putting then on the table
whileinoculating

- The surface of the area is susceptible to microbial contamination due to airborne

contaminants. Holding caps, lids, and cotton plugs instead of leaving them at the
table will prevent the transfer of microorganisms that could interfere with the
expected outcome. This method produces efficiency since extra sterilization steps
will not be needed

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 d. Petri dishes are not fully opened when inoculating or obtaining microbial
specimens.

- The lids of petri dishes are only partially opened when inoculating or obtaining
microbial specimens. If we opened the cover of the petri dish fully, microbes from
the air or any other area of your surroundings would land on the petri dish,
contaminating your experiment. Furthermore, we cannot entirely close the lids
because doing so would prevent oxygen from reaching the bacterium, promoting
the growth of potentially hazardous anaerobic bacteria.

e. Streak dilution using quadrant method

- The steak dilution using quadrant method is the preferred steaking method to
create different areas that will contain varying concetrations of microorganisms
allowing better observation and study of the colony and individual morphology of
the specimen

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 VI. References

Acharya, T.. Streak Plate Methods: Principle, Procedure,Uses. Last updated September 1, 2022, from
https://microbeonline.com/streak-plate-method-principle-purpose-procedure-results/

Aseptic techniques and preparing bacterial plates - Treating, curing and preventing disease - OCR
Gateway - GCSE Biology (Single Science) Revision - OCR Gateway. (n.d.). BBC Bitesize. Retrieved
September 9, 2022, from
https://www.bbc.co.uk/bitesize/guides/z2kvw6f/revision/7

Aseptic techniques. (n.d.). Practical Biology. Retrieved September 9, 2022, from

https://practicalbiology.org/standard-techniques/aseptic-techniques

Medical Microbiology (equipment and procedure). Medical Microbiology (Equipment and Procedure) |
ESSDACK Open Badges. (n.d.). Retrieved September 10, 2022, from
https://badge.essdack.org/badgr/badge_class/194

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