FACULTY OF FOOD SCIENCE AND NUTRITION

NT10502 GENERAL MICROBIOLOGY

PRACTICAL 2: Techniques for Handling Microbial Cultures

GROUP NUMBER:1

DATE (DAY) OF EXPERIMENT:12/11/2018

NAME MATRIX NO. SECTION SIGNATURE

LEE YEK QIUN BN18110044 1
HO QI JING BN18110066 1
NUR ATHIRAH BN18110063 1
HANISAH BT YAAKUB
SYAFAWATI BT AHMAD BN18110058 1
KAMIL
LEE SIOK PEI BN18110109 1
VIVIAN CHONG PIK BN18110048 1
WAN
FELICIA URAI BN18110067 1
BONIFACE
Activity 1

INTRODUCTION

The survival and growth of microorganisms depend on available and a favourable growth
environment. A microbiological culture medium is a substance that encourages the growth,
support, and survival of microorganisms. Culture is the term given to microorganisms that
are cultivated in the lab for the purpose of studying them. Culture media contains nutrients,
growth promoting factors, energy sources, buffer salts, minerals, metals, and gelling agents
(for solid media). There are different types of media suitable for growing different types of
cells. Broth, agar plates and agar slants are the example of the culture medium. Liquid
culture media, commonly called “broth” while solid and semi-solid culture media, commonly
called “agar”. Aseptic technique is a fundamental and important laboratory skill in the field
of microbiology. This technique is for a variety of procedures such as transferring cultures,
inoculating media, isolating of pure cultures and performing microbiological tests. Proper
aseptic technique can greatly minimize or even prevent contamination of cultures from
foreign bacteria inherent in the environment. Furthermore, proper aseptic technique also
can prevent microbes used in the laboratory from accidentally being released into the
environment and infecting people working in the laboratory. This is especially relevant when
pathogens are being handled.

OBJECTIVES

To learn the procedures used in preparing media needed for culturing microorganisms.

Materials and apparatus

Broth media, agar media, laboratory bottle (blue cap), universal tube, petri dish, measuring
cylinder, spatula, glass pipette, magnetic stirring bar, hot plate, Bunsen burner

Procedures

Broth media

1. 3.5g of Yeast Extract-Peptone-Dextrose (YDP) broth media powder was weighed out.
The excess broth media powder that was weighed out is not allowed to return back
into the container.
2. The broth media powder was then dissolved with 100 ml of distilled or deionized
water in a flask. The position of the eyes must be perpendicular to the meniscus of
the measuring cylinder when the measurement is taken.
 3. The solution of broth media was dispensed into universal tube using a graduated
cylinder or a pipette.
4. The screw cap of universal bottle was loosed on.
5. The universal bottle was sterilized at 121℃ for 15 minutes.

Agar media slants

1. The agar powder was weighed out.

2. The distilled water or deionized water was added into a flask. The agar was boiled
and stirred until it was dissolved.
3. 5 ml of molten agar was transferred to each universal tube by using a pipette.
4. The universal tube was sterilized for 121℃ for 15 minutes with the caps was loosed
on.
5. The rack was tilted onto a solid surface while the medium was still hot so that the
medium in the tubes was slanted. The medium was allowed to harden in this
position.
6. The cap was tightened when the medium was cooled.
7. These tubes can be stored at room temperature or in the refrigerator.

Agar media plates

1. The Potato Dextrose Agar (PDA) medium was prepared.

2. The PDA agar was sterilized at 121℃ for 20-25 minutes. Then it was cooled to 50℃.
3. The UV light of lamina air flow was switched on and left for 30 minutes. The UV light
was switched off before the cover was removed after 30 minutes.
4. The surface of working station of lamina air flow was cleaned with 70% of ethanol
before the experiment was conducted.
5. The lid of the bottle was removed and then the neck of the bottle is flamed with
Bunsen burner.
6. The lid of the petri dish was lifted slightly with the other hand and the sterile molten
agar was poured into the petri dish and the lid is replaced.
7. The neck of the bottle was flamed and the lid of the bottle was replaced.
8. The dish was rotated gently to ensure that the medium covered the plate evenly.
The lid of the plate must not touch the agar and the surface of the agar must be
smooth without bubbles.
9. The plate was allowed to solidify.
10. The plate was sealed and incubated in an inverted position.
 Results

Plates 1 2 3 4 5 6 7

Agar thickness 1 1 1 1 1 1 1
3 3 3 3 3 3 3
Presence of × × × × × × ×
agar on lid of
plate

Smoothness √ √ √ √ √ √ ×

Presence of × × × × × × ×
bubble

Presence of × × × × × × ×
contamination

Sterilised broth 1 2 3 4 5 6 7
media
Clarity √ √ √ √ √ √ √
Colour change × × × × × × ×
Presence of × × × × × × ×
bubble
Presence of × × × × × × ×
contamination
Discussion

1. What makes agar surface smooth, no presence of bubbles and no presence of agar
on lid of the plate?

The correct preparation method is followed which the dish is gently rotated to ensure
that the medium covers the plate evenly without touch the lid of the plate and make
sure the surface is smooth with no bubbles

2. How to prevent the presence of contamination on the prepared agar plate?

Agar plate is prepared in lamina air flow which give a clean and sterilize
environment. The cover of petri dish also did not open outside of lamina air flow to
prevent contamination.

3. Why the cap of the universal bottle with the broth media should close loosely but not
tightly?

To ensure that the broth media can sterilize completely in the autoclave.
4. Why all the media solution need to be autoclave at 121 ˚C, 15 psi for 15 minutes?
Autoclave the media at 121 ˚C , 15 psi or 15 minutes is sufficient to ensure complete
destruction of even the spores of bacteria that are able to survive at high
temperature.

Conclusion
All the culture medium are well prepared for the growth of microorganisms such as
broth media, agar slants and agar plate. All the culture medium are in good
conditions such as no bubble, not contaminated, smoothness and the thickness of
1
agar is 3
of the plate.

References
1. National Center for Biotechnology Information. 2012. Aseptic Laboratory
Techniques: Plating Methods. [Online]. In
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846335/

2. University of Alberta. 2018. Autoclave Use and Safety in the Department of

Biological Sciences. [Online]. In
https://www.ualberta.ca/biological-sciences/safety/biosafety/autoclave-safety

3. Virtual Amrita Laboratories Universalizing Education. 2018. Aseptic Technique

and the Transfer of Microorganisms. [Online]. In
http://vlab.amrita.edu/?sub=3&brch=73&sim=212&cnt=1

4. American Pharmaceutical Review. 2014. Assessment of Culture Media in

Pharmaceutical Microbiology. [Online]. In
https://www.americanpharmaceuticalreview.com/Featured-Articles/163589-
Assessment-of-Culture-Media-in-Pharmaceutical-Microbiology/
Activity 2

Introduction

Cultivation is the process of propagating organisms by providing the proper environmental

conditions. Cultivation of microorganisms is very important in microbiological techniques and
is a major task for a microbiologist. It is use for isolating, culturing and identify microorganism.
Therefore, cultivation can be used to isolating microorganism from different environment,
mixed culture and obtained a pure microbe culture. Obtaining a pure culture of bacteria is
usually accomplished by spreading bacteria on the surface of a solid medium so that a single
cell occupies an isolated portion of the agar surface. Pure culture is normally needed for
determination of biological and biochemical activities of the microorganism. Factors that must
be controlled during growth include the nutrients, pH, temperature, aeration, salt
concentration, and ionic strength of the medium. Due to pathogenic potential in certain
microbes, it is very important to avoid contamination and preserve the cultures purity by using
correct handling method.

Objective

1. To investigate the presence of microbes in our skin surface and the air.
2. To study about the amount of microbes present before and after washing hands.
3. To study the difference number of bacterial colony and it morphology from our
own skin surface before and after washing the hand.
4. To study the bacterial colony morphology after the agar plate exposed to air.

Materials

Sterile Plate Count Agar (PCA) media plates

Procedures

1. Two PCA media plates are labelled as before and after at the bottom of the plates.
2. The lid of the plate is lifted slightly and the fingers of the other hand are lightly
touched on the surface of the agar plate and the lid is replaced.
3. Hands are washed with ordinary soap and water and let them to be air dried.
4. Step 2 is repeated with washed hands for the another PCA media plate.
5. The top is removed from the another PCA media and is placed on the bench near the
working station.
6. The agar surface is let to be exposed to the air for 1 hour.
7. All of the PCA media plates are then incubated at 30℃ for 2 days.
 Result:

Plates Set 1 Set 2 Exposed

Before After Before After
Form Punctiform, Punctiform, Punctiform, circular, Punctiform,
Circular, Circular circular punctiform circular,irregular
spindle

Elevation Flat Flat Flat Flat Flat

Margin Entire, Entire, Entire, Entire,undulate Erose,entire,

undulate undulate undulate undulate
Colour Pale yellow, Pale yellow, Pale Pale Pale,creamy
creamy creamy yellow,creamy yellow,creamy yellow
yellow yellow yellow yellow
Optical Translueen Sheen Translueen Sheen Translueen
Properties

Texture Moist Dry Moist Dry Dry

Discussion:
1. Why the agar surface need to be exposed for 1 hour?
To culture microbes from the air and to learn the morphology of colonies that is
present in the air.
2. Why the colonies in the plate before handwash is more than in the plate after
handwash?
Washing hands prevents illnesses and spread of infections to others. Handwashing
with soap removes germs from hands

3. Why is it necessary to sterilize the petri dishes before we start this investigation?
It is important that the cultures are uncontaminated by other microorganisms, so
sterile conditions are needed.
4. Why Plate Count Agar (PCA) is necessary used in this experiment?
PCA agar is a microbiological growth medium commonly used to assess or to monitor
"total" or viable bacterial growth of a sample.
5. Why the both agar plate need to incubate at 30℃ for 2 days after experiment?
This is to reduce the risk of culturing microbes that are pathogens to humans.
Conclusion
The presence of microbes in our skin surface and the air was investigated. The
amount of microbes present before and after washing hands was studied. The
difference number of bacterial colony and it morphology from our own skin surface
before and after washing the hand was studied. The bacterial colony morphology
after the agar plate exposed to air was studied.
References

References activity 2:
1.Cliffsnotes. 2016. Microbial cultivation. [Online]. In
https://www.cliffsnotes.com/study-guides/biology/microbiology/microbial-cultivation-
and-growth/microbial-cultivation

2. Microbiology online. 2018. Observing bacteria in a petri dish. [online]. In

https://microbiologyonline.org/teachers/observing-microbes/observing-bacteria-in-
a-petri-dish

3. Geo F. Brooks, Karen C. Carroll, Janet S. Butel, Stephen A. Morse, Timothy A.

Mietzner. 2013. Jawetz, Melnick, & Adelberg's Medical Microbiology, 26th edition.
New York, NY: McGraw-Hill.
ACTIVITY 3

Introduction

A suitable growth medium must contain all the nutrients required by the microorganism to
be cultivated, and such factors as pH, temperature, and aeration must be carefully
controlled. A liquid medium, broth or the solid medium, agar is normally used to prepare
most of the cultures depending on the purpose of analysis. There are few basic agar or
broth inoculating techniques for culturing and identification purpose. Streaking method is
used to isolate single colony from microbial material. Streak-plate method is used for
isolation to produce isolated colonies of an organism on an agar plate. This is useful when
you need to separate organisms in a mixed culture or when you need to study the colony
morphology of an organism. There are many different streaking patterns for isolating
colonies. After streaking, streaked plate are incubated at 37°C for 24 hours in an inverted
position. This is one of the dilution method uses to grow individual colonies that can be used
as pure culture for further identification tests.

Objective

1. To study about the method to transfer bacterial growth from culture provided to
broth media, surface of agar slope, surface of agar plate.

2. To learn about the technique to isolated single colony from microbial material.

3. To study about the streak plate technique.

Materials and apparatus

3 agar media slope,6 bottle broth media (Activity 1), 6 agar media plate (Activity 1), access
to bacterium growth in broth medium, access to the microbial culture from natural habitats
(Activity 2), Bunsen burner, inoculum wire

Procedures

A. Agar Slants as Sources of Inoculum

1. The universal bottle containing microorganism used were labelled with date, name
and group.
2. The surface of working station was cleaned with 70% of ethanol before the
experiment was conducted.
3. The Bunsen burner was lighted up by using fire igniter.
4. The inoculum wire was sterilized by flaming it to redness with Bunsen burner at the
angle of 45°. The inoculum wire was let to be cool before used.
5. The cap was removed from universal bottle containing microbial culture and the
neck of the bottle was flamed.
6. A small portion of the Micrococcus surface growth was obtained by using the
sterilized inoculum wire. The agar was not allowed to be dig.
7. The cap was removed from universal bottle containing broth media and the neck of
the bottle was flamed.
8. The inoculum wire was immersed in the broth media and the needle was shaken
gently to free the microorganisms sticking to it.
9. The neck of the same bottle was flamed and the cap was replaced.
10. The inoculum wire was flamed again and was put down at suitable place.
11. The broth media was incubated at 30℃.

B. Inoculation of an Agar Slant

1. The agar slant containing the organism used were labelled with date, name and
group.
2. The surface of working station was cleaned with 70% of ethanol before the
experiment was conducted.
3. The Bunsen burner was lighted up by using fire igniter.
4. The inoculum wire was sterilized by flaming it to redness with Bunsen burner at
the angle of 45°. The inoculum wire was let to be cool before used.
5. The cap was removed from universal bottle containing microbial culture and the
neck of the bottle was flamed.
6. A small portion of the Micrococcus surface growth was obtained by using the
sterilized inoculum wire. The agar was not allowed to be dig.
7. The cap was removed from universal bottle containing slant agar and the neck of
the bottle was flamed.
 8. The inoculum wire was placed into sterilized agar slant’s surface at its bottom.
The inoculum wire was moved from side to side as pulled it upward out of the
bottle. The agar was not allowed to be dig.
9. The neck of the same bottle was flamed and the cap was replaced.
10. The inoculum wire was flamed again and was put down at suitable place.
11. The slant agar media was incubated at 30℃

C. Streak Place Technique

1. The bottom of the petri dish was labelled with number of 1,2,3 and 4 as points.
This will make the streaking procedures easier.
2. The surface of working station was cleaned with 70% of ethanol before the
experiment was conducted.
3. The Bunsen burner was lighted up by using fire igniter.
4. The inoculum wire was sterilized by flaming it to redness with Bunsen burner at
the angle of 45°. The inoculum wire was let to be cool before used.
5. The cap was removed from universal bottle containing microbial culture and the
neck of the bottle was flamed.
6. A small portion of the Micrococcus surface growth was obtained by using the
sterilized inoculum wire. The agar was not allowed to be dig.
7. The petri dish was held so that the bottom rests on the palm of the left hand if
right-handed or the right if left-handed.
8. The petri plate cover was lifted and the inoculum was placed at the point labelled
1 of the agar surface. The inoculum was streaked on the agar surface from side
to side in parallel lines to the point labelled “2”.The cover of petri dish was
replaced.
9. The inoculum wire was sterilized by flaming it again.
10. The petri dish was rotated one quarter of full turn.
11. The inoculum wire was cooled and the petri dish cover was lifted at the same
time.
12. The inoculum wire was skimmed over the point labelled “2” and a second set of
streaks was made from point “2” to “3”. The petri dish cover was replaced.
13. Steps 9 to 12 were repeated by streaking from point “3” to “4” and streak
randomly to the middle.
14. The inoculum wire was flamed before putting it down.
15. The petri dish was incubated in inverted position at 30℃.
RESULTS

For the appearance of the broth media:

For broth media, the growth is located at the bottom of the bottle and the rest of the broth
is clear.

For the appearance of agar slants:

For the agar slant, the bacteria grow evenly on it, which called filiform. Filiform means that
bacteria are in dense and opaque growth with a smooth edge.

For the colonies on streak plates:

Agar Agar Agar Agar Agar Agar Agar

Plate 1 Plate 2 Plate 3 Plate 4 Plate 5 Plate 6 Plate 7
Suitable streaking
pattern
Not more than one
incision in the agar √ √ √ √
Presence of at least
10 well separated √ √ √ √
colonies

Absence of aerial √ √ √ √ √ √
contaminates

Recognition of
pure/impure culture √ √ √ √ √ √ √
Recognition of
aerial contaminants

Discussion

1. Why the growth is located at the bottom of the bottle and the rest of the broth is
clear?
This is because the bacteria is obligate anaerobes that can only growth well in the
absence of oxygen. The growth will ceases in the presence of oxygen.

2. Why the bacteria growth will ceases in the presence of oxygen in the broth media
that are obligate anaerobes?
This is because there is lack of enzyme, superoxide dismutase (SOD) or not at all to
neutralize the harmful form of oxygen.

3. How to get or ensure the agar plate have a suitable streaking pattern?
The best results are obtained when the loop is sterilized or flamed between streaks
when using a stainless steel inoculating loop. The plate is turned between streaks.
The first quadrant is a tight streak, and the most growth is here. Density decreases
in the four-streak pattern. Isolation is first obtained in the third or fourth-streak
most of the time. This is where the purest, youngest bacteria are found. Cells from
 the individual, pure colonies may be transferred to sterile media to start pure
cultures. If it is a subculture, test the individual colonies in the last quadrant.

4. Why is it important to avoid digging into the agar with the loop?
The bacteria can grow in the gouge preventing the bacteria from growing into
distinct colonies.

5. What is the purpose of a streak plate?

Technique used to obtain a pure culture of an organism from a mixture of
organisms.

6. Why the aseptic technique is necessary to used in this experiment?

To ensure that the contamination is reduce or eliminate when plating organisms.

Conclusion

The method to transfer bacterial growth from culture provided to broth media, surface of
agar slope, surface of agar plate was studied. The technique to isolated single colony from
microbial material was learned. The streak plate technique was studied.

References