Lab Report: Culturing Microorganism and

Introduction to Microscopy.

Sk. Md. Noor-e-alam.

Group Members:
Chan Ten Kiat
Ishrak Shariar Rafi
Ng Jia Lu
Yew Boon Cheong

School of Engineering
Taylor’s University
Malaysia
21 April 2015

Date of Experiment: 28th November

2015
Report due date: 14th November 2015
Report submission date: 6th December
2015

Checked by:

Item/marks
Format/10
Abstract and
Introduction/10
Figures and Diagrams/15
Materials and Method/10
Results Discussions/45
References/10
Table of Contents

ABSTRACT...............................................................................................................................................1

1.0 Introduction.....................................................................................................................................1-2

2.0 Experiment design...............................................................................................................................2

2.1. Materials..........................................................................................................................................3
2.2 Methods............................................................................................................................................3
2.3. Procedure.....................................................................................................................................3-5

3.0 RESULTS AND DISCUSSIONS....................................................................................................5-9

4.0 ERROR ANALYSIS............................................................................................................................9

5.0 CONCLUSIONS AND RECOMMENDATIONS............................................................................9

REFERENCES........................................................................................................................................10

ABSTRACT
The experiment was carried to prepare pure bacteria culture through streak plate method. This
investigation explains us about the culture medium, that is whether the culure medium is contaniminated
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or polluted by pathogenic microorgamisms or not. The number of living bacteria present in a certain
sample was quantified through viable plate counts method and microorganisms was observed under light
microscope. The highest number of colonies was aroung 221 which was the solution of concentration 10-
5
. And the lowest was 2 for the solution of concentration 10-7. The other one which was the solution of
concentration 10-6 had aroung 33 colonies. All the data were then compared with the theoretical plates.
previously, then after the fact utilizing it to guarantee all microorganisms present on it is executed.

1.0 INTRODUCTION
Streak plate Scientists must be certain that any microbe used or studied in the laboratory has not been
contaminated with others from the environment or from themselves. When working with
microorganisms, it is desirable to start with single, isolated colonies to ensure you are working with a
pure culture. Cultures that are visible on the surface of solid media are called colonies. A colony forms on
a plate when a single microbe is inoculated onto the surface of the plate and reproduces until there are
enough cells to form a visible colony. Since a colony theoretically forms from a single cell, a colony
should then represent a pure culture. One way to obtain single, isolated colonies is using the quadrant
streak method. The quadrant streak plate method allows sequential dilution of the original microbial
material over the entire surface of a fresh plate. As the original sample is diluted by streaking it over
successive quadrants, the number of organisms decreases. Usually by the third or fourth quadrant only a
few organisms are transferred, and these produce single, discrete colonies.
Viable plate count Scientists use a number of different methods to determine the number of micro-
organisms that are present in a given population. This can be accomplished by using the
spectrophotometer to measure the optical density of the population, by directly counting the
microorganisms using a haemocytometer, or by serial diluting the bacteria and plating the diluted bacteria
on media that supports the growth of the micro-organisms. The latter provides statistically accurate and
repeatable results. This method is also the ideal method for enumerating microorganisms in a given
population because it only identifies the living organisms in that population. For example, if one knows
the number of bacteria present in a culture then one can calculate the amount of protein or DNA that can
be isolated from that population. Microbial enumeration is also routinely used in the areas of public
health. In food or water and wastewater industry, food, milk, water are tested for the numbers of
microbial pathogens to determine if these products are safe for human consumption.
Microscopy The light microscope is an important tool in the study of microorganisms. The compound
light microscope uses visible light to directly illuminate specimens in a two lens system, resulting in the
illuminated specimen appearing dark against a bright background. The two lenses present in a compound
microscope are the ocular lens in the eyepiece and the objective lens located in the revolving nosepiece.
Compound light microscopes typically have the following components:
Illuminator: the light source in the base of the microscope.
Condensor: collects and concentrates light from the illuminator and directs it to the iris diaphragm.
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Iris Diaphragm: regulates the amount of light entering the lens system.
Mechanical Stage: a platform used to place the slide on which has a hole in the center to let light from
the illuminator pass through. Often contains stage clips to hold the slide in place.
Body tube: Houses the lens system that magnifies the specimens Upper end of body tube -- Oculars/Eye
pieces: what you view through Lower end of body tube -- Nose-piece: revolves and contains the
objectives

2.0 EXPERIMENT DESIGN

Figure 1: Detail diagram of R713 Refrigeration Laboratory Unit

2.1. Materials
1) Nutrient agar plates,
2) Broth culture,
3) Inoculum loop,
4) Pipettes and pipette tips,
5) Sterile H2O,
6) Hockey stick/ spreader,
7) Glass slide,
8) Glass slide cover slip.
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2.2 Methods
In this experiment the apparatus was first arranged as shown in Figure 1. Four quadrants were prepared.
The sterile inoculum loop in broth culture was touched onto the agar plate in the four quadrat one by
one. After that the plate was sealed and labelled with information such as names etc. The dilution blanks
and agar were then labelled. A sterile pippete was then used to transfer 1 ml of bacterial broth culture
into the tube and was shaken vigorously. Then the solution was diluted and after that the diluted
solutions were spread across the agar plate. All the plates were then sealed and kept under sterile
condition at a temperature of 30oC for around three days. After the plates were compared with the
theoretical plate.

2.3. Procedure
For Streak Plate
1. Into four quadrants divide the agar.
2. Touch the sterile inoculum loop in broth culture and place the loop with culture onto the agar
plate in Quadrant 1 and streak the loop very gently using a back and forth motion.
3. Sterilize the loop.
4. Extend the streaks into a second quadrat (quadrat 2) by going back to the edge of quadrat 1, go
back twice.
5. Again sterilize the loop.
6. Extend the streaks into a second quadrat (quadrat 3) by going back to the edge of quadrat 2, go
back twice.
7. Again sterilize the loop.
8. Extend the streaks into a second quadrat (quadrat 4) by going back to the edge of quadrat 3, go
back twice.
9. But do not go back into quadrant 1.
10. The plate is then closed on both sides and ensure the plate is labeled with name, date and
organisms and incubate it upside down in order to prevent condensation from getting on to agar
at 30 °C.
Viable Plate Count
1. The dilution blanks are then labeled: 10-1, 10-2, 10-3,10-4,10-5,10-6, and 10-7.
2. The agar plates are labeled: 10-5,10-6, and 10-7
3. Using a sterile pipette, transfer 1 ml of the bacteria broth culture into the tube labeled 10-1 and
shake it vigourously.
4. Using a another sterile pipette, transfer 1 ml of the 10-1 tube into the tube labeled 10-2. Again
shake it vigourously.
5. Using a new sterile pipette, transfer 1 ml of the 10-2 tube into the tube labeled 10-3 and mix it
vigourously.
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6. Using a new sterile pipette, transfer 1 ml of the 10-3 tube into the tube labeled 10-4 and mix it
vigourously.
7. Using a new sterile pipette, transfer 1 ml of the 10-4 tube into the tube labeled 10-5 and mix it
vigorously.
8. Using a new sterile pipette, transfer 1 ml of the 10-5 tube into the tube labeled 10-6 and mix it
vigorously.
9. Using a new sterile pipette, transfer 1 ml of the 10-6 tube into the tube labeled 10-7 and mix it
thoroughly.
10. 10.Using a new sterile pipette, transfer 0.1 ml of the 10-7 tube to the nutrient agar plate labeled
10-5, and spread the liquid thoroughly and evenly over the surface of the plate using a sterile
spreader and be careful not to let the spreader dig into the agar.
11. 11.Using a new sterile pipette, transfer 0.1 ml of the 10-6 tube to the nutrient agar plate labeled
10-7 and spread the liquid thoroughly and evenly over the surface of the plate using a sterile
disposable spreader.
12. 12.Using a new sterile pipette, transfer 0.1 ml of the 10-7 tube to the nutrient agar plate labeled
10-8, and spread the liquid thoroughly and evenly over the surface of the plate using a sterile
disposable spreader.
13. 13.After the liquid has absorbed into the plates, tape them closed on both sides. Make sure your
plates are labeled with your name, date, and the organism, and incubate upside down at 30 °C.
Microscopy.
1. Place the slide on the stage and use the coarse and fine focus knobs to bring the specimen into
focus under 4X magnification.
2. Turn the revolving nose piece to bring the 10X objective into place and observe the specimen.
Repeat with the 40X objective. Draw several cells observed under 40X in the spaces provided
below.
3. Turn the revolving nose piece to bring the 100X objective into place and observe the specimen.
Move the nosepiece slightly so you can add a drop of immersion oil to the slide, then bring the oil
immersion objective back into place and fine-tune the focus. Observe the specimen. You may
need to turn down the amount of light in order to observe the specimen under oil immersion and
draw several cells observed under 100X..

3.0 RESULTS AND DISCUSSIONS

Table 1: Tabulated results of the diameter of the indentation of different materials.

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Load, L Pe Pc Tσ T1 T2 T3
(W) (N/m2) (N/m2) (oC) (oC) (oC) (oC)
150 0 800 30.6 -8.6 48.3 28.0
600 130 960 33.4 5.2 62.4 31.3

Discussion:
Streak plate

Figure 2: Streak plate technique results

From the first quadrant to the last quadtrant the microorganism were scattered evenly. In the last
quadrant the colonies were countable due to it lesser number. On the other hand, the colonies in the
diluted solution (10-5) were very difficult to count due its density. The basis of such phenomenom could
be the reason the culture was getting weaker eventually till it reached the end. The microorganisms were
scattered and to grow the microorganisms feeded on the nutrients from the media. Thus the colony had
an independent arrangement. This confirms the presence of the microorganism in a culture known as
broth culture. The growth, colour and shape of the microorganisms were identical meaning that they
were refined.
Viable plate count
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Figure 3: 10-5 diluted solution result

Figure 4: 10-6diluted solution result

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Figure 5: 10-7 diluted solution result

The above figures show the distribution of the colonies. In figure 3 the distribution was most random and
the density was largest. Aprroximately there are 221 colonies refined. Also as the solution got diluted the
microorganisms showed development in the media. From the next two figures (Figure 4 and Figure 5) it
could be observed that the media affected by pollution. And the impurity level in Figure 4 was higher
than impurity level in Figure 5. The colony of organism are very far from segements, which are impure, in
a pertri dish when considering the dilution to be 10-6. Hence this could be a possible reason for such
phenomenon that is observed.
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The next three figures (Figure 6,7 and 8) are showing the regular therotical results for such experiments
conducted. In this it can be observed that the density of the colonies gradually decreased as the
concentration of the solution decreased which in turn decreased the number of microorganisms. When
the two figures (3 and 6) are compared it was observed that the original experimental result colonies
density was slightly lesser than the theoretical results. The reason could be that the in the original
experiment some of the microorganism were dead hence number of colonies reduced. Also in the
theoretical results the colonies were more evenly distrubeted compared to the original data obtained. The
results obtained could be observed to seen to quite similar to the theoretical data.
In this experiment all the parts were in a sterile condition; each time hands were taken in or out of the
apparatus it was washed with sanitisers to maintain the sterile condition. The reason was the
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microorganisms require a sterile condition to culture. If not sanitised then foreign particles or organism
could hamper the experimentation by contamination of the microorganisms causing massive errors in
collection of data. The level of impurity could be determined by using the streak plate and viable plate
count, microorganism which develop from an immaculate culture could be verified by observing the
plates after experimentation and comparing with the therotical data.

3.1 Error Analysis

Counting number of colonies accurately was very difficult for the solution of concentration 10-5. As it
was very dense and great in number so grid system is a possible way to reduce the error but still some
dots miss out.

4.0 Conclusion and Recommendations

Concluding the facts it could be understood that the bacteria could be grow in streak plates, by using
streak plate method. The density of the culture was the highest when the concentration of the solution
was highest and vice versa. The microorganism that grew in the plates could be observed under light
microscope.
Recommendations are to carry out the experiment with cautious as sometimes when hands are taken out
of the apparatus by mistake one forget to use disinfectant before carrying out the experiment again. Also
it is essential to maintain the sterile condition of the laboratory.
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REFERENCES

1. http://www.sciencemag.org/content/296/5570/1127.short
2. Cain, D., Hanks, H., Weis, M., Bottoms, C., and Lawson, J. Microbiology Laboratory Manual. Collin County
Community College District, McKinney, Texas (2013).
3. Robert, A. P., Lorrraine, F., Walter, M., and R.Ronald, M. Laboratory Exercises in Microbiology 2nd Edition. John
Wiley & Sons, Inc., Hoboken, New Jersey (2005).