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FACULTY OF FOOD SCIENCE AND NUTRITION

NT10502 GENERAL MICROBIOLOGY

PRACTICAL 2: TECHNIQUES FOR HANDLING MICROBIAL CULTURES

GROUP NUMBER: 1

DATE (DAY) OF EXPERIMENT: 10.10.2019 (THURSDAY)

NAME MATRIC NO. SECTION

DANIEL VOO CHUNG CHERNG BN19110187 2

CHIN SI NING BN19110146 1

JAZLEE NU'AIM BIN JAIMA'AIN BN19160236 2

ANIS NOREISHA BINTI BN19110025 1

NORAZALAN

NURIN KHAIRINA BINTI BN19110049 1

KAMARUZAMAN

NUR QADIRAH BINTI MOHD SAIPUL BN19110056 1

ANUWAI
INTRODUCTION

Aseptic technique is crucial in characterizing an unknown organism. Oftentimes, multiple

transfer must be made from a stock culture to various test media. It is imperative that only the
desired organism is transferred each time and that no foreign bacteria are introduced during the
transfer. This technique also obligatory in isolating and purifying bacteria from mixed source of
organisms.

This technique ensures that an aseptic environment is maintained when handling

microorganisms. This means that no contaminating microorganisms are introduced into
cultures. Other than that, the microbiologist is not contaminated by cultures that are being
manipulated.

In preparing broth, dissolve it with distilled water until totally dissolved. For the
preparation of agar medium, it needs to do in autoclave so it can sterilize. This because within
a few hours there will be thousands of bacteria reproducing in the media so it has to be
sterilized quickly before the microbes start using the nutrients up.

Pure Culture Technique use in streak plate and pour plate means to isolate an individual
species. Both procedures involve diluting the bacterial cells in a sample to an end point where a
single cell divides giving rise to single pure colony. (Ruangpan L., et al., 2004). Due to
pathogenic potential in certain microbes, the handling method is necessary to preserve the
cultures purity and to avoid contamination. For the streak and pour plates are to separate a
mixed culture of bacteria. The bacteria may be differentiated by the characteristics of the
colony, such as color and shape.
OBJECTIVES

1. To isolate an individual species using streaking technique

2. To observe isolated colony cultured on plate before and after contamination.

MATERIALS AND APPARATUS

Materials

6 Sterile agar media plates

3 agar media slope

6 bottle broth media

Yeast from agar slant

Apparatus

Bunsen burner

Inoculating loop

METHOD

Cultivation of microorganisms from natural habitat

1. 2 petri dishes containing agar media were labeled ‘BEFORE’ and ‘AFTER’ to avoid confusion.

2. The unwashed and dry hand is used to lightly touch the surface of an agar plate in the
‘BEFORE’ labeled petri dish

3. The same hand is washed with ordinary soap and water, air dried and then is used to touch
the surface of an agar plate with the label ‘AFTER’.

4. The lid of the agar plate is removed and the agar plate is placed on the bench with the agar
surface exposed to the air for one hour.
5. The agar plate is then incubated at 30C for 2 days

Cultivation techniques : Transfer and selection of colony

1. The microbial growth of the yeast is transferred from the culture provided to a tube of broth
media.

2. Some bacterial growth from the culture provided are spread over the surface of an agar
slope.

3. Some bacterial growth are streaked out over the surface of an agar plate so as to obtain at
least some well separated colonies on the plate after incubation.

Follow up :

1. The appearance of the broth media and agar slopes cultures are examined and described.

2. The colonies on the streak plates are examined.

• Suitable streaking pattern

• Not more than one incision in the agar

• Presence of at least 10 well separated colonies

• Absence of aerial contaminants

• Recognition of pure/impure culture

• Recognition of aerial contaminants

RESULTS

Activity 1: Preparation of culture media (Broth, Agar Plates and Slants)

Table 1: Observation of culture media that have been prepared

Media Observation

YDP broth Clear brown solution is obtained.

PDA agar Uneven surface of two agar plates due to the

solidification of the molten agar. Presence of
agar on petri dish lid on one of the agar
plates due to excessive swirling. The
remaining three agar plates are in good
condition.

No. for each media prepared (n): 6

Activity 2: Examining colonies on the plates

Table 2: Observation of colonies on plate before wash hand

Type of microbial colonies 1 2 3 4

Colour White Yellow Red White

Form Circular Circular Circular Punctiform

Elevation Flat Raised Raised Flat

Margin Filamentous Entire Filamentous Filamentous

Table 3: Observation of colonies on plate after wash hand

Type of microbial colonies 1 2 3

Colour Cream Cream Yellow

Form Punctiform Circular Circular

Elevation Flat Flat Convex

Margin Entire Entire Entire

Table 4 : Observation of colonies on plate in air

Type of microbial colonies 1 2 3 4 5

Colour White Cream Yellow White White

Form Irregular Circular Circular Circular Irregular

Elevation Umbonate Flat Raised Flat Umbonate

Margin Undulate Entire Undulate Entire Lobate

Activity 3:
A. Observation on inoculated media

Table 5: Observation on broth media

Broth media 1 2 3 4 5 6

Clear solution Yes Yes Yes Yes Yes Yes

Presence of pellicle No No No No No No

Presence of Yes Yes Yes Yes Yes Yes

sediment
Table 6: Observation on agar slope

Agar slope 1 2 3

Presence of filiform pattern Yes Yes Yes

B. Examining colonies on the streak plates

Table 7: Observation of colonies on the streak plate

Plate no. 1 2 3 4 5 6

Streaking pattern 1 1 1 0 1 1

Incision in the agar 0 0 1 0 1 0

Well separated colonies 1 0 1 0 1 1

Aerial contaminants 1 1 1 1 1 1

DISCUSSION

For the preparation of broth media, clear brown solution was obtained and indicates that
there was no contamination. For the preparation of agar media plates, the thickness of agar will
affect the efficiency of the growth of bacteria. So, the thickness of agar should be ¾ of the
plate (Taylor, et al., 2013). If it is too thin, the growth of microorganism will be affected as
there are deficiency of nutrients. Two of our agar media surface were uneven as the liquified
agar has solidified during the preparation steps. The smoothness of agar is important as it will
affect the accuracy when counting the colonies. Observation will be hard if the agar surface is
uneven. Then, there was presence of agar on the lid of one of the agar plates. This is due to
vigorous stirring of plate when trying to ensure even coverage of medium. For the better result,
it should stir it gently as presence of agar on lid may lead to contamination. There were no any
presence of bubbles on our agar plates.
Based on our observation, there are more colonies present on plate after wash hand but
this is supposed to be lesser. This is most probably due to the exposure of air or air flow
through hair dryer during the drying process after hand washing. When dried our hand with the
hair dryer, a gust of air potentially carrying various bacteria had been reintroduced to the
already cleaned hand and thus there are more colonies observed. However, there are lesser
type of microbial colonies identified thus we deduced that the microbial colonies that was not
identified after hand wash are not present in the surrounding air of the laboratory. For the
observation of colonies before wash hand, there was presence of one red and circular colony. It
was predicted as one harmful bacteria which was Serratia marcescens. S.marcescens is a type
of gram-negative bacteria that occurs naturally in soil and water and produce a red pigment at
room temperature (Jane Buckle, 2015). For the observation of colonies after wash hand, there
was the presence of a yellow, circular and convex colony. It was predicted as Staphylococcus
aureus because it fits the cultural characteristics which are circular, convex and entire margin
(Sagar A., 2017). For the agar that was left exposed to air, the agar contained five different
type microbial colonies due to direct exposure to air.
For the culture prepared in liquid broth media, there is presence of sediment and turned
cloudy when broth media was shaken. This indicates that there is growth of microbes inside the
broth and also the culture undergoes anaerobic respiration. This is because the culture grow at
the bottom of the bottles which has low oxygen content. If pellicles are formed instead, the
culture undergoes aerobic respiration. For the culture prepared in solid agar, there is growth of
microbes observed on agar slope. This indicates that the colony has transferred to the culture.
Filiform pattern was observed as yeast cell is a non-motile organism in nature. It is incapable to
propel themselves due to absence of flagella.
Based on our observation for the streak plates, there is one streak plate observed
without streaking pattern as incorrect technique is used. Streaking should be done evenly to
obtain single colonies that can be easily observed. The inoculum loop should be cooled down
properly before streaking another quadrant to prevent denature of colonies due to high
temperature (Pollack, R.A., et al., 2002). There are four streak plates observed with incision in
the agar. The incision occurred as poor technique was applied by application of too much
strength or also can be due to the wire loop was still hot. The incision will form a hole on the
agar surface thus making it difficult to observe the growth of colonies. In handling the streaking
part, make sure they streak with adequate strength and prevent shaky hand during the
streaking process. There are two streak plates observed without well separated colonies. This is
probably because the wire loop was not flame properly (Harrigan, W.F., 1998). There are no
recognition of any aerial contaminants on all of the streak plates.
CONCLUSION

In conclusion, the objectives of this experiment were achieved which are to isolate an
individual species using streaking technique and to observe the isolated colony on plate before
and after contamination. This proven that microbes will live and growth if there is a suitable
conditions that fulfilled its need. Next, to ensure a precise result for this experiment aseptic
technique plays an important role. Always make sure the inoculating loop that use for picks up
culture is sterilised first and give enough time to cool down before picking up culture.

REFERENCES

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Ruangpan, L., & Tendencia, E. A. 2004. Bacterial isolation, identification and storage.
Laboratory manual of standardized methods for antimicrobial sensitivity tests for bacteria
isolated from aquatic animals and environment (pp. 3-11). Aquaculture Department, Southeast
Asian Development Center.

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