College of Nursing

MICROBIOLOGY &
PARASITOLOGY
(Lab)

Experiment No. 7
INOCULATION TECHNIQUES

Group # 5
Group Members CONTRIBUTIONS

GUADILLA, Josana Drawing

GUZMAN, Phoebe Conclusion

HADSAN, Aeryka Jazmine Drawing

LABI, Shandy Hygeia Questions 1-4

LUBEN, Gabriel Kenjie Data and Results

OCSILLOS, Edberg Jann Data and Results

DATA AND RESULTS

INOCULATION TECHNIQUES RESULTS (observations)

a. STREAK PLATE An inoculation of either yeast or bacteria is

gradually diluted over the surface of agar
media that has formed in a Petri dish on a
streak plate. As a result, multiple colonies
on the plate grow far distinct from each
other. The technique's goal is to create
separate, singular, and pure colonies.

b. POUR PLATE In inoculating with the utilization of a pour

plate, after the appropriate sample dilution
has been applied to the plate, the outcome
is colonies that are evenly spread across
the medium.

c. SPREAD PLATE In a spread plate inocculation, the result

would likely have a measurable quantity of
 isolated bacterial colonies dispersed
throughout it.

d. INOCULATING THE AGAR Since the microbes were streaked in the

SLANT FROM THE AGAR agar slant in a serpentine manner, then
PLATE CULTURE after Incubation of the agar slant for 18 to
24 hrs at 37 degrees Celsius, the
organisms grew in the same way we
streaked the agar slant which is in a
serpentine manner.

e. INOCULATING THE AGAR The same result in inoculating the agar

SLANT FROM THE BROTH slant from the agar plate culture but the
CULTURE difference is that we transfer the
microorganism from a very liquidy culture
which is the broth to the agar slant. Again,
since we streak the agar slant in a
serpentine manner, then the growth
pattern of the microorganisms in the agar
slant after incubation is also in a
serpentine pattern.
DRAWING

A. Flame-sterilizing the inoculating loop/needle

B. Streak plate, pour plate and spread plate techniques

C. Technique in inoculating tube cultures
CONCLUSION

A. To perform the various bacterial isolation techniques, namely: streak,

pour plate and spread plate techniques.

STREAK PLATE

1. Sterilize the inoculating loop in the alcohol lamp by clicking on the loop and
dragging it to the flame. Put the loop into the flame until it is red hot. Allow it to
cool.
2. Pick an isolated colony from the agar plate culture and spread it over the first
quadrant (approximately 1/4 of the plate) using close parallel streaks.
3. Flame the loop.
4. Turn the plate 90° and lightly sweep the loop 1-2 times through the inoculated
area, then streak into the next quadrant without overlapping the previous
streaks.
5. Flame the loop.
6. Turn the plate 90°, overlap the previous area 1-2 times, and streak into the
next quadrant as in step 4.
7. Flame the loop.
8. Repeat #6, streaking the remainder of the plate.
9. Invert the plate and incubate at 37 degrees celsius for 18-24 hours

POUR PLATE

1. Put sample from the broth culture into the petri plate/dish
2. Melt agar to 45°C
3. Pour into a Petri dish containing the sample from broth culture
4. Following the addition of the molten-then cooled agar, cover the plate
5. Gently rotate in a circular motion to achieve uniform distribution of
microorganisms.
6. Incubate at 37 degrees celsius for 18-24 hours

SPREAD PLATE

1. Put sample from the broth culture into the agar petri plate/dish
2. Sterilize an L-shaped glass rod by dipping in alcohol and flaming until alcohol
has burned off.
3. Spread the inoculum evenly over the surface of the agar with sterile L-shaped
rod.
4. Incubate at 37 degrees celsius for 18-24 hours.

B. To apply the basic techniques of proper isolation, cultivation and

maintenance of pure cultures of bacteria

In working with microorganisms we must also have a sterile

nutrient-containing medium to grow the organisms. Anything in or on which
we grow a microorganism is termed a medium. A sterile medium is one which
 is free of all life forms. It is usually sterilized by heating it to a temperature
where all contaminating microorganisms are destroyed. Finally, in working
with microorganisms, we must have a method of transferring growing
organisms (called the inoculum) from a pure culture to a sterile medium
without introducing any unwanted outside contaminants. This method of
preventing unwanted microorganisms from gaining access is termed aseptic
technique.

To maintain a pure culture or to obtain a pure culture of a microorganism, a

quadrant/T/isolation/streak plate is performed. The T-streak is a form of
dilutions on a solid surface. In this technique, the plate is divided into sections.
Bacteria are deposited in the first section at full strength from the original
source. Then the inoculating loop is sterilized. From this point on, no
additional cells are added to the agar surface. The sterile loop is used to
spread out cells that have already been placed on the plate. After spreading
cells from the first area to the second, the loop is sterilized again. This
eliminates extra cells from the loop. After all the regions have been
inoculated, the hope is that in the last section cells are far enough apart so
that they grow up into isolated colonies. This technique allows one to observe
isolated colonies and characterize them and determine if your observations
are consistent with our expectations for the organism you are working with.

C. To perform the various methods of inoculating solid or liquid culture

media such as agar plate, agar slant and broth cultures.

SLANT: solid medium made with agar and various nutrients and indicators. Slanting
gives the bacteria a greater surface area on which to grow in a tube. Agar slants are
also useful in maintaining bacterial cultures, more so than stacks of Petri dishes.
Multiple cultures are easily placed into test tube racks and stored under refrigeration.
Bacteria are inoculated onto a slant using a loop and grow in the surface of the agar.
Slant/deeps are inoculated by stabbing a needle into the butt and then immediately
streaking across the surface of the slant.

PLATE: solid medium made with agar and various nutrients and indicators. Can be
made in Petri dishes of various sizes. Plates are particularly helpful in isolating a
specific species of bacteria, which is not possible in a liquid medium. Using the SFIC
technique bacteria can be diluted until individual colonies are formed. Bacteria are
inoculated onto a plate using a loop.
Inoculating a Plate from a Broth Culture

1. Sterilize the inoculating loop.

2. Remove the cap from tube. Do NOT put the cap of the tube down on the lab
bench—hold it in your hand.
3. Flame the lip of the tube.
4. Place sterile portion of inoculating loop into broth, then remove.
5. Flame the lip of the tube
6. Replace the cap.
7. Gently streak the surface of an agar plate with the inoculating loop.
8. Sterilize the inoculating loop.
QUESTIONS FOR RESEARCH

1. What is the purpose of flame-sterilizing the inoculating loop/needle

before and after using it?
Any microorganisms on the loop that may contaminate the culture are destroyed by
flaming before use. Any microorganisms left on the loop from the bacterial transfer
activities are destroyed by flaming after usage. This makes sure that contaminated
bacterial spores are eliminated. Also, blazing the inoculating loop sterilizes it and
permits the dilution of the inoculum. Less organisms can be streaked in each region,
resulting in the desired isolation or separation of colonies. Lastly, when the loop has
been sterilized, never set it down otherwise it might get infected once more.

2. How does the distribution of growth on a pour plate different from that
on a streak plate?
The primary distinction between a pour plate and a streak plate is that in a pour
plate, the bacterial broth is added first, followed by the nutrient agar, whereas in a
streak plate, the melted nutrient agar is poured first and a loop of bacteria from a
slant is added second. Moreover, the amount of inoculum in the streak plate is only a
loopful from a bacterial slant, but the amount in the pour plate is between 1.0 and 0.1
mL. Furthermore, the streak plate is used to isolate colonies, whilst the pour plate is
used to count the quantity of colonies.

3. Why is aseptic bacteriologic technique so important in the isolation of

pure cultures?
The danger of contamination can be considerably decreased or minimized by using
the right aseptic method. In transferring cultures onto fresh medium, aseptic method
frequently preserves pure stock cultures and single spore cultures. Proper aseptic
practices guard against the unintended introduction of microorganisms into the
environment and/or the contamination of lab users. Moreover, using proper aseptic
technique has avoided environmental contamination of the cultures from both inborn
and outborn bacteria. Using the surveyor's health, the lab benchtop, unsterilized
glassware and equipment, dust, and other regions as an example, airborne bacteria
(such as fungus) might interfere with an experiment's ability to provide accurate data.

4. What is meant by “streak dilution” technique?

The streak plate technique is based on the dilution theory. One way to characterize it
is as a quick qualitative isolation method. Having fewer colonies is the key
requirement for isolation. In this method, a loopful of culture is put over an agar plate
to acquire the necessary distance between individual cells. The inoculum is gradually
diluted via the streaking technique, allowing the bacteria to be counted as colony
forming units. Using regions of increasing dilution on a single plate, the streak plate's
goal is to produce isolated colonies from an inoculum. Since they were created from
a single progenitor cell, isolated colonies are a clone of cells.
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