BASIC MICROBIOLOGY PRACTICUM

OBSERVATION PURE CULTURE AND STAINING AT BACTERIA AND FUNGI

NAME : DIANA WITA EKASARI SIHOTANG

ID NUMBER : 4173342002
CLASS : BILINGUAL BIOLOGY EDUCATION 2017
DEPARTMENT : BIOLOGY
PROGRAME : BIOLOGY EDUCATION
GROUP : IV ( FOURTH )
EXP.DATA : SEPT,27th 2019

BIOLOGICAL DEPARTMENT

FACULTY OF MATH AND SCIENCE

 MEDAN STATE UNIVERSITY

Y.A.2019/2020

I. TITLE : THE PURE CULTURE ISOLATION OBSERVATION AND STAINING

AT BACTERIA

II. THE AIM : 1.TO KNOW WHAT IS PURE CULTURE

2.TO KNOW WHAT IS STAINING TECHNIC
3.TO KNOW WHAT KIND OF BACTERIA THAT WAS USED
4. TO OBTAIN A SINGLE COLONY ON MICROBIAL PURIFICATION.
5. TO DIFFERENTIATE BACTERIAL SPECIES INTO 2 LARGE GROUP
NAMELY GRAM POSITIVE AND GRAM NEGATIVE BASED ON THE
CHEMICAL AND PHYSICAL PROPERTIES OF THE CELL WALL

III.THEORITICAL REVIEW
Isolation is a way to separate or remove certain microbes from the environment, so that pure
culture or pure culture is obtained. Pure culture is a culture in which microbial cells originate
from the division of a single cell (Pelczar, 1986). Pure culture or pure culture is very useful in
microbiology, which is to study and identify microorganisms, including the study of cultural,
morphological, physiological, and serological characteristics, requiring a population consisting
of only one type of microorganism (Hadioetomo, 1993). The nature of the organism in a pure
culture can be studied with very hard methods with very accurate results because the influence of
other living cells can be nullified (Volk, 1993).
There are several ways to prevent the entry of undesirable microorganisms and to plant a species,
namely: Planting by scratching, is a routine method used to isolate microbes to obtain pure
culture. Field planting is carried out by wetting the entire surface of the agar plate with a
microbial suspension. There are several ways to get pure culture, namely: dilution, pouring,
scraping, single cell isolation, animal inoculation (Budiarti, 2009).
Some factors that need to be considered in carrying out microbial isolation are: the nature of each
type of microbe to be isolated, the place of life or origin of the microbe, the appropriate growth
medium, how to inoculate microbes, how to incubate microbes, how to test that the isolated
microbes have been in the form of pure culture and in accordance with what is meant, how to
maintain that the microbes that have been isolated remain pure culture.
The use of media is not only for microbial growth and propagation, but also for other purposes,
for example for isolation, selection, evaluation, and differentiation obtained. This means that the
use of certain types of substances that have an influence on the growth and propagation of
microbes, is mostly done and used. So that each media has its own characteristics (specifications)
according to its purpose (Baker, 1986).
IV.APPARATUS AND MATERIALS
3.2.1 Tools
The tools used in this lab are sterile petri dishes, labels, glass objects, cover glass, bunsen, ose
needles, and microscopes.
3.2.2 Material
The ingredients used are 96% alcohol, 70% alcohol distilled water, NA medium, violet crystals,
lugol, safranin, isolates (Klapsiella & Escherichia coli).
V.WORK PROCEDURE
1. Preparation
Before doing this practicum, do it with aseptic technique, namely, the workplace is sterilized
with 70% alcohol and lit bunsen so that the lab work is more aseptic.
2 Purification of Microbes
Prepared petri dishes containing solid media (certain types of media). After that, a colony of
microorganisms from the selected culture was taken with a sterile ointment needle measuring 2
mm and the ose needle carefully etched on the solid media. Then, every move from 1st to 2nd,
3rd and so on the ose needle must be burned on top of Bunsen. Then, the petri dish is incubated
and then purified by taking 1 colony from the petri dish using a sterile ointment needle
measuring 2 mm and finally prepared a test tube containing a slanted media then the ose needle
was etched on the surface so that it is carefully tilted and placed in an incubator.
3. Bacterial Staining
Prepared tools and materials used during this practicum. After that, the glass preparations and
object glass are taken. Then, the microbes that are taken from the bacteria used and chopped on
the glass preparations. After that, the glass preparation containing the bacteria was fixed above
the Bunsen to dry. Then, take 2-4 drops of violet crystals and let stand for 1 minute. Then, rinse
with distilled water. Next, the preparations were given lugol which was dropped for 1 minute and
rinsed with distilled water. After that, the glass preparation was given 96% alcohol which was
 dropped, allowed to stand for 30 seconds and rinsed with distilled water.
After that, the glass preparation was given safranin for 20 seconds, allowed
to stand and rinsed with distilled water. Finally, the glass preparation is
covered with slide and observed under a microscope.
4. Observations
After purifying and staining the gram, gram purification is observed
whether or not many microbes have been etched on the medium. And in gram staining, bacteria
are used.

No. Gambar Keterangan

1.

Media that will used as nutrition of

bactery

2.

The result of pure culture

3.

The result of pure culture isolate after

24 hours.

VI.RESULT
At the pure culture we get result namely :
Table result of staining and pure culture

Species Type of grouwth Picture

Klapsiella pneunominiae

No. Aspek yang diamati Keterangan

1. Bentuk koloni monococcus, diplococcus, dan streptococcus
2. Bentuk sel Coccus
3. Sifat gram bakteri Negatif
VII.DISCUSSION
FACTORS AFFECTING GROWTH
 BACTERIA
Growth is defined as an increase in all chemical constituents cell. This is a process that requires
full replication structure, organelles, and cellular protoplasmic components in the presence of
internal nutrients the surrounding environment. In bacterial growth, all essential substances must
be available for the synthesis and maintenance of protoplasms, with energy sources,and suitable
environmental conditions.As a group, bacteria are very "smart" organisms.They show a very
large ability to use materials scattered food, arranging inorganic materials into organic
compounds that are very complex. Some species also learn to grow in various niches ecological
with extreme temperature, acidity and oxygen pressure.The ability of bacteria to survive under
such ambient conditions is a protection from high adaptability and reflex capacity in success
responds to a stimulus that is considered unfamiliar or never encountere previous.

1. Nutrition Factors
Carbon. Two basic patterns of bacterial nutrition need and mirror ability Its metabolism is
presented in Table 4-2. Autotrophic bacteria (lithotroph), for its growth only requires water,
inorganic salt and carbon dioxide. This group synthesizes carbon dioxide to a large extent
essential organic metabolites. Heterotrophic bacteria (organotrophs) need carbon organic for its
growth. In laboratory practice, glucose is broad used as an organic carbon source, but various
other compounds can also be used specifically or certain carbon sources by different bacteria.
 Growth Factor.
A number of heterorophic bacteria cannot grow without supply one or more growth factors. The
compound is usually added in culture medium in the form of yeast extract or blood, including vitamin B-
complex,amino acids, purines, and pyrimidines. Vitamin B-complex acts as a catalyticin cells are also
components of coenzymes or as a prosthetic group of enzymes. Organism which is able to synthesize
growth factors usually does not require compounds from the outside.
Inorganic ions.
A small amount of inorganic ion is needed by all bacteria.In addition to nitrogen, sulfur and phosphorus
are contained as elements in compounds biological function, potassium, magnesium and calcium in
bacteria related functions with certain anionic polymers. Magnesium functions to stabilize ribosomes,
cell membranes, nucleic acids, and are needed for the activity of a number of enzymes. Potassium also
needed for the activity of a number of enzymes, and the concentration of potassium in cells Gram-
positive bacteria are affected by the content of the teichoic acid in cell walls.Most bacteria need iron,
magnesium, zinc, copper, and cobalt, and for other bacteria the need for molybdenum and selenium is
considered essential.The need for these elements for other bacteria is more difficult to estimate, because
sometimes necessary or its presence is considered as an element of contaminants in the medium.
Elements in small amounts (trace elements) play an important role in parasitic host contraction. In
animal hosts, the strength of iron-binding proteins inside body fluids function to hold iron against
microorganisms that attack enter. The success of microorganisms entering the host, will be able to
increase its ability to take iron, and actively extract iron from various environments. A number of iron
compounds (siderophore) are already known in several species of bacteria. Its presence is very important
for iron taking, and significant evolutionarily for the success of the competition with its deep host
limited amount of essential nutrients.
Oxygen.
The oxygen demand in certain bacteria reflects the mechanism which is used to meet its energy needs.
Based on need oxygen, bacteria can be separated into five groups:
1. Obligate anaerobes that grow only in a state of oxygen pressure
very low and oxygen is toxic.
2. Anaerobic aerotolerants that are not killed by oxygen exposure.
3. Facultative anaerobic, can grow in aerobic and anaerobic conditions.
4. Obligate aerobes, need oxygen for growth.
5. Microaerophilic bacteria that grow well at low oxygen pressure,
oxygen pressure height can inhibit growth.
In anaerobic tolerant and obligate, its metabolism is strong fermentative.
In facultative anaerobes, the way metabolism of respiration is done if oxygen is available,
but does not occur fermentation. At the time the bacteria grow in a state
air, a number of enzymatic reactions occur and result in hydrogen production
peroxide and superoxide radicals. In aerobic, aerotolerant, and anaerobic bacteria
Facultatively, the superoxide dismutase enzyme prevents the accumulation of superoxide ions the obligate
anaerobic enzymes are not present:

In facultative and aerobic anaerobic bacteria, hydrogen peroxide formed in the dismutase reaction are
rapidly destroyed by catalase. Although Aerotolerant bacteria, such as lactic acid bacteria do not have
catalase, peroxidase it has can damage H2O2, causing bacteria to grow on the state of oxygen
availability.Targets that might be damaged by H2O2 and O2 - including membrane proteins beyond
specific, redox active components on the cytoplasmic membrane, and enzymes on periplasma area. In
Treponema pallidum, oxygen sensitivity becomes relative against DNA damage caused by H2O2.
Carbon dioxide.
Bacteria that use CO2 as a source of cellular carbon main, are chemolithotrophic and photolithotrophic
bacteria. In addition, kemoorganotrof also requires an adequate supply of CO2 for heterotrophic and for
CO2 fixation fatty acid synthesis. Carbon dioxide is normally produced during catabolism organic
compounds, therefore not considered a limiting factor. Some bacteria, such as Neisseria and Brucella,
have one or many enzymes low affinity for CO2 and requires CO2 at more concentrations high (10%)
compared to CO2 found in the atmosphere (0.03%). This situation must be considered for the sake of
isolation and culture of these bacteria.
2. Physical Factors
Reduction-Oxidation Potential. Reduction-Oxidation Potential (Eh) on the medium
culture is a critical factor in determining the growth of an existing inoculum
when transferred to new media. In most contact media with air,Eh around + 0.2 to + 0.4 Volts at pH 7.
Obligate anaerobes cannot grow in such circumstances, Eh is needed at least - 0.2 Volts. Situation
Anaerobic culture can be made by releasing oxygen, using a system anaerobic culture or by the addition
of sulfidril-containing compounds,like calcium thioglycate (mercaptoacetate). During its growth aerobic
bacteria and anaerobic decreased environmental Eh, this can be observed and important in purulent
infections caused by a mixture of aerobic bacteria and anaerobes that can cause infections that are started
by aerobic bacteria.Temperature Each bacterium has the optimal temperature at which they are can grow
very fast and have a temperature range where they can
grow. Cell division is very sensitive to the effects of damage caused
the temperature; large and strange calves can be observed in the growth of culture on
temperatures higher than temperatures that support growth rates
very fast.
Based on the temperature range where growth can occur, bacteria
grouped into three:
1. Psychophilic, -5oC to 30oC, optimum at 10-20oC;
2. Mesophilic, 10-45oC, optimum at 20-40oC;
3. Thermophilic, 25-80oC, optimum at 50-60oC.
The optimal temperature usually reflects the normal environment
microorganisms. So, pathogenic bacteria in humans usually grow well on
temperature of 37oC.

Hydrogen Ion Concentration.

The pH of the culture medium also affects
growth speed, for bacterial growth there is also a range of pH and pH
optimal. In pathogenic bacteria the optimal pH is 7.2 - 7.6. Although the medium is on
initially conditioned with the pH needed for growth but, in a manner
the gradual magnitude of growth will be limited by the metabolite products produced
the microorganism.
Bacteria have a very effective mechanism for maintaining control regulation of cytoplasmic pH (pHi).
In some bacteria, the pH differs from 0.1 unit per change in pH at external pH. This is due to the control
of system activity transpr ion which facilitates the entry of protons. Various systems reflect the wide
range of pH values shown by various bacteria. Acidophilic
has a pH range of 6.5 - 7.0; neutrophils have a pH range of 7.5 - 8.0,and alkalophils have a pH range of
8.4 - 9.0. Fermentative microorganisms shows a higher pHi range than microorganisms that use the
respiratory pathway. In microorganisms fermentative, acidic fermentative product production and
accumulation resulting in impaired pH balance and growth restriction. A number microorganisms
increase the compensation mechanism to prevent toxic effects from the accumulation of acidic and
highly concentrated products. Example this mechanism, by inducing new metabolic pathways for
production purposes
neutral butanol product from butyrate by Clostridium acetobutylicum and butanediol
from acetate by Klebsiella aerogenes.
Osmotic Conditions.
The concentration of an osmotically active solution in the cell bacteria, generally higher than outside
cell concentrations. Mostly bacteria,except for Mycoplasma and bacteria that have cell dinging
damage,intolerant of osmotic changes and will develop a transport system
complex and osmotic sensor-regulating devices to maintain the osmotic state
constants in cells.
Membrane-derived Oligosaccharide (MDO), a cell element that is present on E. coli. In E. coli and
other Gram-negative bacteria, there are two parts to the liquid which is different, the cytoplasm
contained in the inner membrane, and the periplasma region which is between the outer membrane and
the inner membrane. At this time of bacteria grows o medium with low osmolarity hence the cytoplasmic
membrane a little stiff will expand at least can prevent changes in osmolarity periplasma area, the same
as in the cytoplasm.In cells that grow deep medium with low osmolarity, MDO is the main source of
anions fixed in the periplasma area and plays a role in maintaining high osmotic pressure and Donnan
membrane potential in the periplasm section. This oligosaccharide structure very appropriate for the role
of the arrangement. This oligosaccharide has an intermediate B2200-2600 and is impermeable to the
outer membrane, a component important for its specific function. These oligosaccharides consist of 8-10
glucose units.Cell growth in a medium with low osmolarity synthesizes MDO on maximum speed, the
speed of synthesis seems to be genetically regulated for respond to changes in osmolarity of the medium.
The function Media Mining at media in pure bacterial cultures
As a source of food

The reason
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