LABORATORY REPORT

PRACTICAL 5

FUNDAMENTAL MICROBIOLOGY (ADS 3113)

NAME : NURUL AINAAMIRA BT MOHD NOR RASHIDI

MATRIX NO : 025694

PRACTICAL : MICROSCOPE: USE, CARE AND OBSERVATION OF PREPARED

MICROORGANISM SLIDES

LECTURER : PROFESSOR DR. MUHAJIR HAMID

DEMONSTRATOR : EN.AHMAD LUTFI BIN LUQMAN

DATE OF EXPERIMENT: 2 AUGUST 2009

DATE OF SUBMIT : 9 AUGUST 2009

INTRODUCTION

Microscopy is the technology of making very small things visible to the human eye. Basically,
microbiology are related to microorganism which are very small and tiny and only can be seen
through the microscope which are various of types and functions. Microscope is an instrument
which is first invented by Anton van Leeuwenhoek and undergoes evolution from time to time
but retain the same function to observe microorganisms. Usually we use the unit to measure
microorganism like micrometer, nanometer and angstrom. There are many types of microscopes
such as simple microscope, compound light microscope, dark-field, phase-contrast, fluorescent
microscopes, electron microscopes divided into two: transmission e.m and scanning e.m and
confocal microscope and also digital microscope. Briefly, simple microscope use visible light to
observed of dead stained organism or live one with sufficient natural color contrast. Dark- field

OBJECTIVES

1. To learn the techniques to prepare media or culture medium for microorganisms or

microbes
2. To recognizes the functions of various media prepared
3. To identify the importance of sterilization, heating and freezing of media
4. To differentiate the types of media and its forms

MATERIALS

Distilled water, Nutrient Agar powder for plate (20 g/L,4 g/200 ml ), Nutrient Agar powder for
slant ( 20 g/L, 2 g/100 ml ) Mueller-Hinton agar powder (34 g/L, 6.8 g/200 ml ). Nutrient broth
powder ( 13 g/L, 1.3 g/100 ml )

METHODS

1. Preparation for Mueller-Hinton Agar

Weight 6.8 g of Mueller-Hinton Agar powder to prepare for 200 ml

Mix the MHA powder and distilled water in the beaker until the amount reached 200 ml

Put the stirrer into the beaker and stir until all the powder dissolved completely
 Labeled the beaker with indicator tape and ready to autoclave at 121 degree C for 15
minutes and at pressure of 15 psi

After autoclave, dispense the 200 ml MHA into sterile plastic petri dish and let it
solidified and cover the plate and stored in chiller at 2-8 degree C before usage

2. Preparation for Nutrient Agar plate

Weight 4 g of Nutrient Agar powder to prepare in an amount of 200 ml

Mix the powder with distilled water until the amount reached 200 ml in a conical flask

Stir until it completely dissolved and autoclave them

After we have autoclave the medium, dispense them in sterile plastic petri dish and let it
solidified and cover the plate

Keep the media in the chiller at 2-8 degree C before usage

3. Preparation for Nutrient Agar slant

Weight 2 g of NA powder to prepare 100 ml of this media

 Mix with distilled water in a beaker until reached amount of 100 ml

Stir and heat them to make sure the powder is completely dissolved and melted on the
hot plate stirrer

Dispense the media in culture tube in slant position until it solidified and capped the
tubes and autoclave at 121 degree C, for 15 mins and 15 psi and stored in chiller before
use

4. Preparation for Nutrient Broth Agar

Weight 1.3 g of NB powder to prepare 100 ml of this media

Mix with distilled water in a conical flusk until the volume reached 100 ml

Stir and heat so that it can completely dissolved and melted on the hot plate stirrer, after
that dispense into culture tube and ready to autoclave, lastly keep in the chiller before use

RESULT

Media Volume Plate/Tube Notes

Mueller-Hinton agar 200 ml plate Get 8 plates
Nutrient agar plate 200 ml plate Get 11 plates
Nutrient agar slant 100 ml tube Get 11 out of 15 tubes
Nutrient Broth 100 ml tube Get 12 tubes
DISCUSSION

For Nutrient Agar slant, we got 11 tubes out of 15, thats mean 4 tubes failed to be slant media.
There are some probabilities due to this failure, if we heat the media and completely dissolved
but quite late to dispense into the culture tube, thus the agar will cover the bottom of the media
and there will be not enough agar in the culture tube. After that, why we must autoclave the
media at 121 degree Celcius for 15 minutes and pressure of 15 psi? It is because in the laboratory
we have fixed the temperature, pressure and time so that we can destroy the bacteria, fungi and
viruses on the media. If the temperature, pressure and time exceeded the true requirement, there
is possability where all the nutrients of the media will eliminated and not suitable for the culture
medium. Same will happened when the requirement temperature, pressure and time is less,
maybe the bacteria will not destroyed. For TCBS agar ( Thiosulfate Citrate Bile Sucrose Agar )
or blood agar, we do not have to autoclave the media instead of heat them because the media will
hemolize. Blood is sterilized aseptically when its purchased meaning animal is killing a certain
way to ensure this happened. So, the blood in our blood agar plate has already been aseptically
sterilized. Last but not least, why we must keep the agar plate in chiller at 2-8 degree Celcius for
storage before usage because to retard the growth of microorganism and to ensure the
contamination process not occur.

CONCLUSION

From the experiment I know how to prepare my own media with the right techniques. Besides
that, actually there are various types of media or culture medium with have its own function and
exists in different forms. On top of that, sterilization through the process of autoclave is very
important for culture medium in order to eliminate bacteria, viruses or even spores contained in
the media that had been prepared.

QUESTIONS

1. How are peptone, beef, and yeast extracts produced? What are the specific nutrients
produced by these compounds? What nutrients do these extracts provide to the
microorganisms?

Peptone are produced from cooking milk/ meat products in acid, most are made by
incubating milk/ meat with trypsin, pepsin or other proteolytic enzymes to digest protein
to a mixture of amino acids, peptides, polypeptides. Peptone produced nitrogen and
provides them for the microorganism. Beef extracts is prepared by concentrating in
 vacuum kettles the clear broth obtained from cooking beef flesh. Beef extracts can
produces vitamins, minerals and nutrients. Yeast extracts obtained from the release of
peptides, amino acids, vitamins and other yeast cell components which once the insoluble
components have been removed. Yeast extracts provide vitamins, minerals, and digested
nucleic acids and also vitamin B-complex and dextransucrase.

2. What is the source of agar? What are the chemical properties of agar and the functions of
agar in the culture medium? At what temperature does agar liquefy/solidify?

Agar is a gelatinous substance derived from seaweed or Ceylon moss. Agar is a type of
gelling agent, clarifying agent and is a polymer, its made up of subunits of sugar
galactose. Agar is a solid substrate that can solidify and contained in culture medium for
microbiological work and provide a solid surface containing medium for the growth of
bacteria and fungi. Agar liquefy at 85 degree Celcius ( 358 K, 185 F ) and solidify at 32-
40 degree Celcius ( 305-313 K, 90-104 F ).

3. What the purpose of the sterilization of the media? How the sterilization of media can be
done?

The purpose of sterilization of media is to inactivate all fungi, bacteria, viruses, and also
bacterial spores be quite resistant. Heat sterilization or autoclave or also known as
converter is done by fixed the temperature and pressure for a a specific time (121 degree
Celcius for 15 minutes and 15 psi ). Before autoclave, we must put the indicator tape so
that when autoclave, chemical in the tape will change color when appropriate conditions
have been met.
 4. Name another five media with its main ingredient and functions

Media Main ingredient Functions

eosin-methylene blue agar Methylene blue toxic to Gram-positive
(EMB) bacteria, allowing only the
growth of Gram negative
bacteria
Lysogeny Broth agar Carbon sources such as Used for the maintenance and
glycerol or glucose and propagation of Escherichia
nitrogen source such as coli (E.coli)
ammonium salts or nitrates
MacConkey Agar Lactose and sucrose For use as a selective and
differential medium for the
isolation of gram negative
bacilli (including coliform
organisms and enteric
pathogens) on the basis of
lactose fermentation
Blood Agar bouvine heart blood used for the growth of only
select microorganisms and in
cell culture to ensure the
survival or proliferation of
cells with certain properties,
such as antibiotic resistance
or the ability to synthesize a
certain metabolite.
Chocolate Agar heat-treated blood turns brown and gives the
medium the color for which it
is named.

REFERENCES

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2. Wikipedia.com
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environmental microbiology". The Microbial World. Retrieved on 2007-07-23.
5. Jacquelyn G. Black , Microbiology seventh edition, Wiley Publisher.