OBJECTIVE

1. To obtained the basic skill of aseptic technique in the field of microbiology.

2. To avoid contamination of cultures and media from microbes in the environment.

3. To transfer cultures from one medium by inoculating to another medium.

RESULTS
A) Inoculation of bacteria (Escherichia coli- E. coli)

i) Plate 1 - Inoculation of bacteria onto solid culture medium (Nutrient Agar/NA) and
streaking plate (Quadrant streak plate) by using the aseptic technique.

Figure 1: Quadrant streak plate after an overnight

Observation: There was a growth of E. coli and there
were a few single colony of E. coli spotted.
 ii) Plate 2 – Inoculation of plate using the simplified streaking technique (zig-zag
streaking).

Figure 2: Zig-zag streaking

Observation: There was a growth of E. coli

B) Inoculation of bacteria in a liquid cultured medium Nutrient Broth and streak on

slant agar.

Figure 3: Nutrient Broth after 1 hour

Observation: Nutrient Broth turns turbid.
 Figure 5: Slant agar after overnight.
Observation: There is a few colony of E. coli on slant agar

Figure 6: Streak agar after overnight.

Observation: A hole that is making on the slant agar become larger.
DISCUSSION
A (i) Plate 1:– Inoculation of bacteria onto solid culture medium (Nutrient Agar/NA) and
streaking plate (Quadrant streak plate).

From the observation, we can see that there is the growth of E. coli in the solid culture

medium because the presence of whitish spots on the solid medium culture after the solid

medium culture is left overnight in the incubator with 37⁰C. The petri dish was invert when in

the incubator in order to avoid the condensation droplets will not fall on the agar’s surface as the

water droplets are potential sources of contamination. Besides that, we also can see the different

concentration growth of E. coli in different quadrant. The first quadrant has the most

concentration of E. coli followed by the second quadrant. At the third quadrant the concentration

of E. coli is less than the second quadrant while at the forth quadrant we can see that there are a

few single colony of E. coli spotted. The quadrant streaking and aseptic technique is used to

inoculate E. coli from one solid agar medium contains E. coli bacteria to the solid agar medium

that does not contain any bacteria. Each quadrant must be overlapped with each other in order to

spread the E. coli on the agar nutrient so that we can observe the growth of the bacteria in the

colonies. In our inoculation process there is no contamination occurred as we wear the mask

during inoculation process to avoid the airborne contamination and we also heating the

inoculation loop before streak on the plate contain the E. coli which can reduce the

contamination before streaking it on the solid agar medium. We also need to cool down the

inoculation loop before streak on the plate contain E. coli to prevent from kill the E. coli before

transfer into the new plate containing solid culture media. These techniques are important in

order isolate a particular type of bacteria from a clinical sample. Streak plate technique is used to

grow bacteria on a growth media surface so that individual bacterial colonies are isolated and

sampled. When the selected culture media is inoculated using a single isolated colony, the
resulting culture grows from that selected single clone.

(ii) Plate 2: – Inoculation of plate using the simplified streaking technique (zigzag

streaking).

In the plate 2, we can see the whitish spots also appeared on the plate after left the plate

in the incubator for 24 hours with 37⁰C but with the different pattern compare to the plate 1. That

indicates there is the growth of E. coli in the plate 2. We also use the streaking technique and

aseptic technique to inoculate the E .coli from plate that contains E. coli to the plate 2 that

contains solid culture media without any bacteria. The aseptic technique is the practices and

procedures to prevent contamination from pathogens such as the wearing the mask during the

inoculation process, heating the inoculation loop before streak on the plate contains E. coli and

spray the workplace with 70% of ethanol and dry it for a while before make the inoculation

process. The 70% ethanol was used because the 70% of ethanol is the most effective in killing

microbe, if the concentration of ethanol is 90%, it will evaporate fast and not be much effective.

The zigzag streaking technique is started with the streaking on the plate that contained E. coli

and then make the streaking the short straight vertical line followed by the zigzag pattern on the

plate that contained solid culture media without any microorganism. The purpose of making

short vertical line by using inoculum loop on the agar is to make sure the E. coli distributed

equally on the medium by ‘zigzag’ streaking. This is called dilution process. If the short vertical

line is not being done at the beginning, the E. coli will not be distributed evenly and most of the

E. coli will grow at the initial part of ‘zigzag’ streaking.

B. Inoculation of bacteria in a liquid cultured medium Nutrient Broth (NB) and streak –
slant agar.

In this experiment, the inoculation of E. coli from the solid culture medium contains E.

coli to the Nutrient Broth. After a colony of E. coli was transfer to the Nutrient Broth. The

Nutrient Broth is left in the incubator for 1- hour period with temperature (37⁰C) and the change

was observed. The Nutrient Broth turns cloudy after 1-hour period in the incubator. The turbidity

shows that the E. coli managed to grow in Nutrient Broth with the optimum temperature. The E.

coli in the Nutrient Broth were transferred to the streak- slant agar and slant agar by using

inoculum loop.

After an overnight, there are growths of E. coli on the surface of streak-slant agar and

slant agar as there the presence of white spotted on the streak slant agar. There also only E. coli

that are grow on the slant agar as there is no other microorganism are present on the slant agar.

This is happened because before streaking the agar in parallel starting at the edge of agar, the

wire loop is sterilized on flame makes the other microorganism die on the inoculation loop

before transfer the E. coli from the Nutrient Broth to the slant agar. Besides that, the incubating

period of streak-slant agar inside the incubator is more than 24 hours. In addition, the growth of

E. coli is fully mature and grown. The purpose of streak-slant agar is to increase the life of

bacteria for research on future.

There are many precautious steps that students need to take note. Small amount of inoculum

should be taken from the cultured E. coli so that colonies of E. coli can be formed easily.

Nutrient Broth (NB), the plate agar and streak-slant agar must be incubated in an incubator with

warm temperature (37oC) because this condition is really suitable for the E. coli to grow.

Besides, the agar plate and streak-slant agar must be incubated within 24 hours so that E. coli can
have sufficient time for growth. The plates must be incubated upside down to prevent the

moisture in the nutrient agar plate from being evaporated on the lid of the plate. Besides that, the

inoculation process must be done near the heat source to prevent the contaminant of the culture

media.
Summary

The aseptic and streaking technique is important in medical and pharmacy field in order

to identify the characteristic of specific bacteria by only inoculating one bacteria species only. It

is important to ensure that the environment during the inoculation process is sterile to avoid other

than desire bacteria from contaminate the culture. For example, when a doctor take a specimen

from their patients, it is important to ensure that only desire bacteria is growth in order to know

what is the suitable antibiotic to give to the patient. In this practical also, there a lot of technique

need to be mastered in order to do inoculation process of E. coli such as heating the inoculation

loop before transfer the E. coli the culture medium, clean the workplace by spray the 70% of

ethanol in order to avoid the growth of the other microorganism. The quadrant streaking

technique also helps to produce the bacteria in the form of single colony. If the all the precaution

steps are being followed, there are no such contaminant to the medium culture and there is no

growth of other microorganism. The importance of aseptic technique in pharmaceutical industry

setting is essential in the making of sterile pharmaceutical products. For instance, the production

of antibiotic uses aseptic technique because the culture bacteria are needed for testing of

antibiotic. Speed, accuracy and economy movement are very important features in order to get

good aseptic technique. Researchers and workers of pharmaceutical industry must be well

trained, motivated, and familiar with the task in hand so that good result of research can be

obtained. If the aseptic technique is poor, contamination of microbes will occur and it will affect

the results of the experiment and life.

Reference

Boylston, A. (2012). The origins of inoculation. Journal of the Royal Society of Medicine,

105(7), 309–313. http://doi.org/10.1258/jrsm.2012.12k044

Fleming, Diane O. 1995. Chapter 13 in Laboratory Safety-Principles and Practices, 2nd Ed.

Edited by Diane O. Fleming, John H.

Koneman, Elmer W., Stephen D. Allen, William M. Janda, Paul C. Schreckenberger, and

Washington C. Winn, Jr. 1997. Chapter 2 in Color Atlas and Textbook of Diagnostic

Microbiology, 5th Ed. Lippincott-Raven Publishers, Philadelphia, PA.

Richardson, Jerry J. Tulis, and Donald Vesley.American Society for Microbiology, Washington,

DC.