THE MANUFACTURE OF MEDIUM AND STERILIZATION

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Name : Hasnadhiazahra Rohadi

SID : B1B015028
Group :5
Subgroup :I
Asisstant : Wahyu Dwi Saputra

MYCOLOGY PRACTICAL WORK REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2018
 I. INTRODUCTION

A. Background

Mushrooms are non chlorophyllic organisms that have four traits namely,
heterotrop, saprophyte, mutualistic and parasitic (Suparti et al., 2016). A fungus is
any member of the group of eukaryotic organisms that includes microorganisms such
as yeasts and molds, as well as the more familiar mushrooms. These organisms are
classified as a kingdom, Fungi, which is separate from the other eukaryotic life
kingdoms of plants and animals. A characteristic that places fungi in a different
kingdom from plants, bacteria and some protists, is chitin in their cell walls. Similar
to animals, fungi are heterotrophs; they acquire their food by absorbing dissolved
molecules, typically by secreting digestive enzymes into their environment. Fungi do
not photosynthesize. Growth is their means of mobility, except for spores (a few of
which are flagellated), which may travel through the air or water. Fungi are the
principal decomposers in ecological systems (Fardiaz, 1992).
Microorganisms are almost omnipresent, very diverse and indispensable to
human survival. Preparation of suitable culture media is one of the prerequisites to
study them. Different microorganisms thrive at different environments and have
variety of growth requirements; like nutrients, pH, osmotic conditions and
temperature. Media containing high carbohydrate source, nitrogen source are
required for the growth of fungi at pH range of 5 to 6, and a temperature range from
15 to 37˚C. There are two general types of fungal culture media: natural and
synthetic. Natural media are composed of natural substrates, such as herbaceous or
woody stems, seeds, leaves, corn meal, wheat germ, and oatmeal etc. Natural media
are usually easy to prepare but they have the disadvantage of their unknown
composition. Some examples include corn meal agar, potato dextrose agar, V-8 juice
agar, and dung agar. Synthetic media, on the other hand, contain ingredients of
known composition (Basu et al., 2015).
Microorganisms can be grown and developed on a substrate called a medium.
With the growth medium, microbial activity can be studied, the microbial isolation
with pure cultures can be done, propagation, testing physiological properties, and
also the number calculation of microbes can be done. The wide diversity in the
nutrients types for microbes is have to be matched by the availability of various
media for the cultivation. The media used such as peptone, meat extract, yeast
extract, and agar. The most common materials used to make the solid medium is agar
(Sutedjo, 1991).

B. Objective
The objective of the manufacture of medium and sterilization practical
activity are :
1. To know the manufactures of fungi medium and various sterilization
 II. LITERATURE REVIEW

Fungi are eukaryotic, spore producing, non chlorophylous organisms with

absorptive nutrition that generally reproduce both sexually and asexually, and whose
usually filamentous, branched, somatic structure, known as hyphae, typically are
surrounded by cell wall (Alexopoulos & Mims, 1979). They are heterotrophic and
depends upon organic matter for nutrition and consequently lives saprophytic or
parasitically or symbiotically on or with other organisms (Kaul, 1997). Fungi are the
agent responsible for much of the disintegration of organic matter and as such they
affect as directly by destroying food, fabrics, letter, and other consumer, good
manufactured from raw material subject to fungal attack (Winterhoff, 1992). Fungi
are a very large, diverse group of living organisms found in nearly all ecosystems.
they have microscopic organelles within their cells called nuclei which contain
genetic material in the form of thread-like chromosomes, enabling hereditary
characters to be passed on to subsequent generations (Rimbawanto & Beedle, 2006).
A growth medium or culture medium is a solid or liquid or semi-solid
designed to support the growth of microorganism or cells or small plants like
the moss Physcomitrella patens. Different types of media are used for growing
different types of cells. The two major types of growth media are those used for cell
culture, which use specific cell types derived from plants or animals,
and microbiological culture, which are used for growing microorganisms, such
as bacteria or fungi. The most common growth media for microorganisms are
nutrient broths and agar plates; specialized media are sometimes required for
microorganism and cell culture growth (Madigan, 2005).
The survival and growth of microorganisms depend on the availability of
nutrients and favorable growth environment. In the laboratory, the nutritional
preparations used to grow microorganisms are called medium (Presscott, 2002).
Media can be classified by their shape, chemical order, and function. Based on the
shape consists of solid media, semi-solid media, and liquid media. The basic nutrient
which needed by microorganisms is divided into seven namely: water, energy source,
carbon source, electron acceptor source, a mineral source, growth factor, and
nitrogen source (Pujiati, 2012).
Sterilization (or sterilisation) refers to any process that eliminates, removes,
kills, or deactivates all forms of life and other biological agents (such
as fungi, bacteria, viruses, spore forms, prions, unicellular eukaryotic organisms such
as Plasmodium, etc.) present in a specified region, such as a surface, a volume of
fluid, medication, or in a compound such as biological culture media. Sterilization
can be achieved through various means, including: heat, chemicals, irradiation, high
pressure, and filtration. Sterilization is distinct from disinfection, sanitization,
and pasteurization, in that sterilization kills, deactivates, or eliminates all forms of
life and other biological agents which are present (Raju, 1993).
B. Discussion

Growth medium is a place that contains various components of nutrients for

growing microorganisms or microbes. Growth medium must meet the requirements
of the nutrients needed by a microorganisms. Nutrition which is needed by
microorganisms for growth are carbon, nitrogen, non-metallic elements such as
sulfur and phosphorus and metals elements such as Ca, Zn, Na, K, Cu, Mn, Fe,
Vitamins, Water, and energy (Anisah & Triastuti, 2015). A growth
medium or culture medium is a solid or liquid or semi-solid designed to support the
growth of microorganism or cells or small plants like the moss Physcomitrella
patens. Different types of media are used for growing different types of cells. The
two major types of growth media are those used for cell culture, which use specific
cell types derived from plants or animals, and microbiological culture, which are
used for growing microorganisms, such as bacteria or fungi. The most common
growth media for microorganisms are nutrient broths and agar plates; specialized
media are sometimes required for microorganism and cell culture growth (Madigan,
2005).
Microbial culture media can be of different type, depending on the nutritional
growth requirements of the microorganisms. Microorganisms require about 10
macroelements namely (C, O, H, N, S, P, K, Ca, Mg and Fe). The first six
components are used in the synthesis of Carbohydrates, Lipids, Proteins and Nucleic
acids and the remaining four exist in the cell as cations and play a variety of roles. In
addition to macroelements, all microorganisms require several microelements like
(Mn, Zn, Co, Mo, Ni and Cu). These are generally part of enzymes and cofactors.
Microorganisms also require growth factors, which are organic compounds (Basu et
al., 2015).
Basic materials in the manufacture of growth medium are solvent, compactor,
and nutrition. Solvent used to condense the growth medium such as agar, gelatin,
silica gel. Agar is the best compactor because there are several microorganisms that
can not decompose. Gelatin is a compactor for all the microorganisms that can
decompose. And silica gel as a compactor which is used to provide nutrients for the
organisms. The example of material that must be included into a medium that used as
a nutrient are carbon that obtained from carbohydrates, nitrogen from ammonia and
urea, elements of metallic and non metallic minerals and vitamins (Anisah &
Triastuti, 2015). According to Madigan (2005), the growth medium of
microorganisms must meet the following requirements namely: contains all the
nutrients needed for the growth and development of microorganisms, have an
optimum osmotic pressure, optimum humidity, and pH in accordance with the needs
of microbes. The media must be in a sterile state, meaning before planting the
microorganisms, not overgrown by other unexpected microbes.
According to Kuntarti (2008), growth media has a function including for media
isolation, for purification, cultivation, and to observe the morphological character of
the microorganisms. There are various kinds of growth media of the organism, some
of them are namely:
1. Nutrient Agar (NA)
Nutrient agar is a common medium for water testing, and dairy products.
Nutrient agar (NA) is majorly used for the growth of microorganism which is not
selective or heterotrophic microorganisms. This media is a simple medium which
made from beef extract, peptone, and agar. Nutrient agar (NA) is a medium that
commonly used in the procedure of bacteriological test of water quality, sewage,
food product, to growth the sample for bacteria test, and for microorganism isolation.
The peptone added to nutrient agar (NA) in order for increase the growth of the
microorganisms (Dwidjoseputro, 2005).
2. Potato Dextrose Agar (PDA)
Potato dextrose agar or PDA is a medium which were made in this lab
activity. Potato Dextrose Agar is used for the cultivation of fungi in a laboratory
setting. Potato Dextrose Agar is not intended for use in the diagnosis of disease or
other conditions in humans (Faddin, 1985). Potato extract is a source of
carbohydrates as the nutritional nutrients for the fungi. Dextrose as the source of
carbon, agar as the compactor, and water as the solvent. PDA media has a main
composition of 200 grams of potato, 20 gram of agar or compactor, 20 gram of
dextrose, and distilledwater of 1000 ml (Stainer, 2011). Those composition is the
same composition used in the lab activity for per 1L of PDA medium manufactures.
According to Japanese Pharmacopoeia (2007), for the commercially PDA medium,
added 39 grams of commercial PDA medium powder per 1 L distilled water. The
composition of the commercially PDA medium per liter is the same namely, potato
200 grams, dextrose 20 grams, and agar 15 grams. Also according to Paul et al.
(2015) Potato Dextrose Agar (PDA) medium plates at 25°C were used to grown the
fungi cultures. Then after the sufficient growth, fresh cultures are used for further
process. Composition for per liter of PDA was Potato 200g, Dextrose-20g, Agar 15g.
3. Yeast Malt Medium
Yeast malt extract agar was the second media which was made in the lab
activity. According to Mcginnis et al. (1982), yeast malt extract agar is a solid
medium recommended for use in qualitative procedures for cultivation and
enumeration of yeast and molds. Malt media for yeast and molds have been used for
many years. The yeast malt extract agar is prepared according to the formulation of
wickerham. Yeast malt extract agar consist of peptone which is the source of carbon,
nitrogen and amino acid. Malt extract supplies nutrient necessary for the growth of
fungi. Dextrose is a carbon energy source and yeast extract supplies B-complex
vitamin to stimulates the growth of yeast and molds. Composition of yeast malt
extract agar per liter was dextrose 10 grams, peptone 5 g, and malt extract 3 g. the
pH of the medium is 6.2 (it is adjusted as required to meet the performance standard)
and the optimal incubation temperature is in between 25-30°C (Haley & Callaway,
1978).
According to Sumanti (2008), the growth medium based on the physical
properties are divided into three namely:
1. A solid medium, the medium which contain 15% of agar so that after it is getting
cold the media will be a solid media. For example, Nutrient agar (NA) and TSA
2. The semi solid media, is a medium which contain 0.3%-0.4% of agar so that it
become more resilient, not solid and not liquid. Semi solid media created with the
purpose so the growing microbes can spread throughout the media but did not
experience the perfect mix if its shaken. For example, bacteria that grow on NFB.
Semi solid medium also aims to prevent or suppress diffusion of oxygen, for
example, nitrate broth medium.
3. The liquid medium, is a medium which does not contain agar or 0% of agar. For
example, NB (nutrient broth).
According to Frobisher (1974), the growth medium based on its shape is divided
into three, namely:
1. Upright form, it has a volume of media for 5-8 ml and it’s a less aseptic. The
method is poured it after it was sterilized and it is used to test the motility and
morphology of the microorganisms.
2. Slant shape, it has a volume of media for 3-5 ml, it’s less aseptic. The method is
poured it first then sterilize it. The function of slant medium is for stock.
3. Petri dish, it has a volume of agar for 8-10 ml and it’s aseptic. The method is
sterilized it first then poured it to the petri dish. The function is to observe the
morphological in yeast and bacteria.
According to Hans (1993), based on the composition of the growth medium, the
growth medium can be divided into three, namely:
1. The natural medium (non-Synthetic) it is a medium that composed of natural
ingredients which the composition can not be known certainty and are usually
extracted directly from the basic materials. For example : tomato juice agar.
2. The semi Synthetic media, is a media that is composed of natural ingredients and
also chemicals ingredients so the composition is not fully known. For example:
potato dextrose agar (PDA).
3. Synthetic media is a media which is composed of chemical compunds and the
ingredients is measured or fully known, for example: macconkey agar.
According to stainer (2011), media growth based of its function or purpose is
divided into three there are
1. Common medium, is a medium which containing basic needs and supporting the
growth of certain microorganisms. For example: NA and PDA
2. Selective medium, is a medium which used to grow certain microorganisms or
the medium which added by inhibitors. For example: singella salmonella agar
(SSA).
3. Differential medium, is a medium which used to distinguish between types of
microorganisms that have a close genetic relationship. For example: ENDO and
EMBA.
Sterilization is defined as the process where all the living microorganisms,
including bacterial or fungal spores are killed from their environment. The purpose
of sterilization is to destroy all pathogenic living microorganisms. Sterilization in
microbiological works like preparation of culture media, reagents, and the equipment
where a sterile condition has to be maintained. Sterilization can be done in three
ways namely: physical, mechanical, and chemical. There are thing that must be
considered for the selection of sterilization methods namely, the toxicity of the
materials which will be sterilized, the stability or the material composition, the
affectivity of the sterilization method used, the efficiency, the benefit obtained using
certain types of sterilization and the last is the time (Hadioetomo, 1993).
According to Hadioetomo (1993), Sterilization divided into three namely:
1. Chemical sterilization
It is typically uses a disinfectant or antiseptic compounds. For example, alcohol.
Disinfectant used to kill microorganisms on inanimate surfaces such as glass and
desks. Antiseptic used to kill microbes in tissue or living thing, for example skin
(Hamdan, 2012).
2. Mechanic sterilization
The mechanic sterilization can be done with filtration, namely sterilization with
milipore filter, this process intended for sterilized the materials which sensitive to
heat such as protein, enzymes, antibiotic substances and etc, The size of the milipore
filter for bacteria is 0,22 µm and for yeast is 0,45 µm (Hamdan, 2012).
3. Physical sterilization
Physical sterilization can be done by heating or irradiation namely,
 Direct fire or heating, it is burned directly with fire. Usually for tools made of
metal like tweezers and ose needle.
 Dry heat, sterilization by using oven with temperature approximately 160oC -
180 oC. This sterilization is suitable for tools made of glass, for example,
erlenmayer flask, test tube, Petri dish, and etc.
 Steam sterilization, is similar with steaming concepts. Material which contain
high moisture content was sterilized using this method to avoid dehydration.
For example, Arnold steam sterilizer.
 Pressurized steam, is sterilization using autoclave with the temperature of 121
o
C, 2 atm pressure and duration of 15 minutes for materials and 20 minutes
for tools (Hadioetomo, 1993).
 V. CONCLUCION AND SUGGESTION

A. Conclusion

Based on the result and discussion above, can be concluded :

1. The manufactures of fungi medium in this lab activity were using PDA medium
which composed of 200 g of potato, 15 g of agar and 20 g of dextrose, 1000 ml
of distilled water. And the yeast malt medium which composed of distilled
water 1000 ml, glucose 10 g, pepton 1 g, extract alt 3 g, extract yeast 5 g. There
are several types of sterilization namely, mechanical which used Millipore
filtration, chemical which used disinfectant and physical.

B. Suggestion

The suggestion for this practical activity are the madia manufactures
simulation should be clearly described so the student could understand fully of how
the medium manufactures was. And for the sterilization there have to be
demonstration of how to use various type of sterilization method.

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