The isolation of the culture by the silicone soap barrier

and methylcellulose gel is not only important because of

the storage factor but also insures that after inoculation
of soil therewith, there will not be a sudden dilution.
To elaborate, the emulsification and break through of the
barrier occurs gradually in the soil and thus the bacteria
in the bark particle can thrive within the particle after
activation and accomplish breakdown of the particle to
humus thereby providing additional nutrients in the soil.
Furthermore, the cultivation media and microorganisms
in the particle will attract respective organisms already
present in the soil and thereby stimulate microbial activity.

Multimicrobial inoculation has been proposed as a way of protecting plants

against environmental stress and increasing the sustainability of plant
production. To study these possibilities in a micropropagation system,
microplants of strawberry, Fragaria × ananssa, were inoculated or left
uninoculated with five microorganisms (Glomus mosseae BEG29, Bacillus
subtilis M3, Trichoderma harzianum DB11, Pseudomonas fluorescens C7r12
and Gliocladium catenulatum Gliomix®), used either singly or in dual mixtures
in the presence or absence of the strawberry diseases crown rot (Phytophthora
cactorum) and red stele (P. fragariae). Finnish light Sphagnum peat was used as
the growth substrate in the experiments. Seven experiments were performed as
two to three months pot experiments in greenhouses of research laboratories in
Finland and Belgium and in a nursery in Finland. In most experiments, the
inoculated microorganims were detected at sufficient densities four weeks after
inoculation. Exceptions were T. harzianum and G. mosseae which were detected
at insufficient densities in several experiments. This might have been due to the
biological and/or nutritional properties of the peat. None of the microorganisms
or their mixtures caused significant growth-promoting effects in more than two
experiments. Dual inoculation did not increase growth more than inoculation
with single organisms. B. subtilis was the most promising growth promoting
microorganism. Most of the microbial treatments decreased crown rot shoot
symptoms as well as the numbers of oospores in the roots when the experiment
was performed in autumn. In the summer experiment with conditions more
favourable for strawberry growth, no disease control was obtained, but some of
the microorganisms increased the severity of crown rot. No microbial treatment
decreased shoot symptoms of red stele, but the degree of root necrosis was
slightly decreased by B. subtilis and G. mosseae + G. catenulatum. The numbers
of oospores of P. fragariae in strawberry roots were not decreased by any
treatment, but several treatments increased them. Both growth promotion and
disease control considered, the single microorganisms T. harzianum, G.
catenulatum and B. subtilis as well as the mixture T. harzianum + G.
catenulatum were the most promising treatments in this study. However, the
great variation between experiments indicates that more studies are needed for
optimization of the whole plant–substrate–microorganism system. The
importance of microbial inoculation for ensuring subsequent growth in the field
also needs to be studied.
https://www.sciencedirect.com/science/article/pii/S0929139304000782

2 Inoculation of culture media All culture media must be checked visually before use for
contamination, significant physical imperfections (for example, uneven distribution of media,
variable amounts of medium in petri dishes/tubes/bottles, colour, gross deformation of the
surface on the media) and expiry date. Culture media should have an identifiable batch or quality
control number and have passed QC tests before use. Plates that are beyond their expiry date,
contaminated plates, and broth media appearing unusually turbid should be discarded4 . For the
effective detection of the bacterial content of specimens, it is important to achieve growth of
individual colonies by using a good technique to inoculate the specimen on culture media. There
are many variations and personal preferences for “plating out”, some of which are described in
this document. The initial area inoculated should cover between a quarter and a third of the total
area of agar used (Figure 1). Whole plates, half plates, or quarter plates can be used depending on
the circumstances (Figures 2, 3 and 4). Specimens may be plated out for individual colonies, or
seeded directly over an entire segment of a plate and incubated without further spreading.
Antimicrobial discs for identification (for example, optochin, bacitracin) may be added as
appropriate. Discs should be placed near the edge of the plate, between the areas covered by the
first and second spread, to avoid total inhibition of very susceptible organisms (see figures 2 and
3). Inoculation loops are designed for quantitative procedures such as sampling, serial dilutions,
as well as for bacterial inoculation. Inoculation loops can be ‘wire or disposable loops’. Disposable
loops were initially used in safety cabinets to avoid sterilisation with Bunsen burners but now
their use is common practice to comply with the health and safety regulations. Disposable loops
are also desirable for quantitative purposes. Wire loops are rarely used in clinical microbiology
laboratories in the UK to reduce the risk of infection from aerosols of pathogenic organisms and,
crosscontamination from improper sterilisation of the wire loops. Therefore, disposable loops are
recommended in this document. For polymicrobial clinical specimens, the disposable loop should
either be changed between each series of streaks, or the loop may be rotated to make the next
series of streaks with the unused side of the loop. For semi-quantitative analysis of urine, the loop
should be changed between streaks. All media should be incubated as soon as possible after
inoculation. In particular, plates for anaerobic incubation should be incubated as soon as possible
to prevent loss of viability

https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/
file/583859/Q_5i2.pdf
Inoculation validation The main dilemma when performing microbiology challenge testing, is the
selection and validation of a method of inoculation. The bacterial strains selected for challenge
testing should always be associated with the product being tested, ideally by being natural
isolates, e.g. from earlier outbreaks or strains isolated from similar products, similarly processed.
Preparation of the bacterial inoculum is also very important; strains should be prepared in
carefully selected conditions so stress on entering the new environment, is minimized. The
method of inoculation must always be validated and must not change the product structure or
chemical or physical characteristics, e.g. water activity, pH, gas within the packaging. etc. To
achieve a satisfactory inoculation, many methods have been developed and are in general use:
direct inoculation, using micro-pipettes are generally used for liquid samples where bacteria are
dispersed in the tested product: spraying techniques, dispersing bacteria on the surface of the
product can be used: MAP or vacuum-packed products, are usually inoculated using syringe and
needle injecting through inoculation tape or inoculating opened packs and re-packing after
inoculation: products of low water activity, where addition of liquid phase is not acceptable, are
inoculated using glass beads covered with biofilm, or silicon sand, chalk or product dust may be
used as bacteria carriers; these latter methods are often used for inoculation of powders, seeds
and nut kernels, transferring bacteria into the product or dispersing them on the product surface.
The number and distribution of the inoculated cells must be determined before proceeding with
the challenge test. If Optical Density is read, the number of cells can be read either directly from
the graph or by simple calculation from the curve equation. For example, the equation for E.coli y
= 1E+09x4 - 2E+09x3 + 2E+09x2 + 6E+08x + 3E+07 can be transformed to Excel formula:
y=1E+09*POWER(x,4)2E+9*POWER(x,3)+2E+09*POWER(x,2)+6E+08*x+3E+07 and the number of
cells will be automatically calculated. Inoculation techniques in Microbiology Challenge Testing
Grzegorz Rachon MSc y = 3E+08x 3 + 4E+08x 2 + 1E+09x + 1E+07 R² = 0.9998 y = 1E+09x 4 - 2E+09x
3 + 2E+09x 2 + 6E+08x + 3E+07 R² = 0.9997 y = 2E+08x 2 + 4E+07x + 2E+07 R² = 0.9517 y = 3E+08x
2 + 5E+08x - 3E+06 R² = 0.9997 y = 3E+08x 3 - 1E+08x 2 + 5E+08x + 282875 R² = 0.9995 0.00E+00
1.00E+08 2.00E+08 3.00E+08 4.00E+08 5.00E+08 6.00E+08 7.00E+08 8.00E+08 9.00E+08 1.00E+09
1.10E+09 1.20E+09 1.30E+09 1.40E+09 1.50E+09 1.60E+09 1.70E+09 1.80E+09 1.90E+09 2.00E+09
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95
1.00 1.05 1.10 1.15 1.20 cfu/g OD Optical density of bacterial suspensions at 350 nm Salmonella
E.coli Staph Cocci Lactobacilli Level of Inoculation The level of inoculation is also very important;
too high a number of bacteria added to the product can interfere with the natural microflora of
the tested product and therefore cannot be used, and too low initial levels may not survive an
adaptation phase after inoculation. Thus, the number of viable cells in the washed and ready-to-
use microbial cocktail is critical and must be obtained prior to inoculation. Since cell cocktails used
for challenge testing should always be made on the day of inoculation, enumeration using
alternative rapid methods, is essential. Only one exception from this rule is acceptable that refers
to bacterial spores which are quite stable when stored in chilled conditions and can be prepared
and enumerated several days before inoculation, using standard viable count techniques.
Microscopic rapid enumerations of yeast or mould spore cocktails can be performed using
counting chambers (Fuchs-Rosenthal chamber) and is widely used and always gives satisfactory
results compared with results obtained from viable count techniques; only one limitation of this
method is size of microorganisms as small cells can be easily missed during counting and
contribute to large reading errors. For small bacteria (Salmonella, E.coli, Listeria, Lactobacillus,
etc.) rapid enumeration of cells using optical density measurement (OD) has been introduced and
is used in Leatherhead on a daily basis. In this method the Optical Density of a cocktail and its
dilutions is measured and the number of cells is calculated from earlier prepared concentration
curves of OD vs. Cell Concentration (cfu/mL)

file:///C:/Users/HP/Downloads/InoculationTechniquesinMicrobiologyChallengeTesting.pdf
Student Preparation for

laboratory Sessions

The efficient performance of laboratory exercises

mandates that you attend each session fully

prepared to execute the required procedures.

Read the assigned experimental protocols to

effectively plan and organize the related activi-

ties. This will allow you to maximize use of

laboratory time.

Preparation of

experimental Materials

Microscope Slides: Meticulously clean

slides are essential for microscopic work.

Commercially precleaned slides should be used

for each microscopic slide preparation. However,

wipe these slides with dry lens paper to remove

dust and finger marks prior to their use. With a

glassware marking pencil, label one end of each

slide with the abbreviated name of the organism

to be viewed.

Labeling of Culture Vessels: Generally,

microbiological experiments require the use

of a number of different test organisms and

a variety of culture media. To ensure the suc-

cessful completion of experiments, organize all

experimental cultures and sterile media at the

start of each experiment. Label culture vessels

with non–water-soluble glassware markers and/

or self-stick labels prior to their inoculation.

The labeling on each of the experimental ves-

sels should include the name of the test organ-

ism, the name of the medium, the dilution of

sample (if any), your name or initials, and the

date. Place labeling directly below the cap of the

culture tube. When labeling Petri dish cultures,

only the name of the organism(s) should be

written on the bottom of the plate, close to its

periphery, to prevent obscuring observation of

the results. The additional information for the

identification of the culture should be written

on the cover of the Petri dish.

inoculation Procedures

Aseptic techniques for the transfer or isolation

of microorganisms, using the necessary transfer

instruments, are described fully in the experi-

ments in Part 1 of the manual. Technical skill

will be acquired through repetitive practice.

Inoculating Loops and Needles: It is impera-

tive that you incinerate the entire wire to ensure

absolute sterilization. The shaft should also be

briefly passed through the flame to remove any

dust or possible contaminants. To avoid killing

the cells and splattering the culture, cool the

inoculating wire by tapping the inner surface of

the culture tube or the Petri dish cover prior to

obtaining the inoculum, or touch the edge of the

medium in the plate.

When performing an aseptic transfer of mi-

croorganisms, a minute amount of inoculum is

required. If an agar culture is used, touch only a

single area of growth with the inoculating wire to

obtain the inoculum. Never drag the loop or needle

over the entire surface, and take care not to dig into

the solid medium. If a broth medium is used, first

tap the bottom of the tube against the palm of your

hand to suspend the microorganisms. Caution: Do

not tap the culture vigorously as this may cause

spills or excessive foaming of the culture, which

may denature the proteins in the medium.

Pipettes: Use only sterile, disposable pipettes

or glass pipettes sterilized in a canister. The prac-

tice of pipetting by mouth has been discontinued

to eliminate the possibility of autoinfection by

accidentally imbibing the culture or infectious

body fluids. Instead, use a mechanical pipetting

device to obtain and deliver the material to be

inoculated.

INOKULASI MIKROBA
Andi Nur Fajri Suloi1), Irma Kamaruddin2)
Abstrak
Penanaman mikroba atau biasa juga disebut inokulasi adalah suatu proses
memindahkan bakteri dari medium yang lama ke medium yang baru dengan tingkat
ketelitian yang sangat tinggi. Pada praktikum inokulasi mikroba, ada dua metode yang
digunakan untuk menumbuhkan mikroba, yaitu metode pour plate dan metode spread plate.
Metode pour plate menumbuhkan mikroba yang suspensinya berada di antara media.
Metode spread plate menumbuhkan mikroba yang suspensinya berada di permukaan yang
diratakan menggunakan drugalsky agar pertumbuhan tersebar merata. Proses inokulasi
diawali dengan pengenceran bertingkat pada suspensi. Pengenceran bertingkat bertujuan
untuk mengurangi jumlah mikroba yang ada di dalam suspensi sehingga jumlah koloni
dalam media mudah dihitung. Pengenceran bertingkat menggunakan larutan fisiologis yang
bertujuan untuk menjaga tekanan osmotik antara cairan di dalam sel dengan cairan di luar
sel agar sel tidak terjadi lisis. Proses inokulasi diakhiri dengan inkubasi inokulum untuk
pertumbuhan optimum mikroba. Inkubasi adalah proses pemeraman media kultur pada suhu
tertentu. Alat yang digunakan untuk proses inkubasi disebut inkubator.
Kata Kunci : Inokulasi, Pengenceran Bertingkat, Pour Plate, Spread Plate
 I. PENDAHULUAN
I.1 Latar Belakang
Tomat (Solanum lycopersicum) merupakan salah satu tanaman yang sangat dikenal oleh
masyarakat Indonesia. Dalam kehidupan sehari-hari tomat memegang peranan penting, sebagai
sumber vitamin C sehingga memiliki banyak manfaat bagi kesehatan dan memiliki potensi
pemasaran yang tinggi. Masalah utama dalam produksi dan pemasaran buah dan sayuran segar
adalah aspek mutu dan keamanan pangan, dimana buah tomat memiliki daya tahan yang rendah
terhadap penyimpanan yang lama dan mudah busuk. Pembusukan pada buah tomat diakibatkan
oleh bakteri Erwinia carotovora yang menyebabkan buah hancur, berair dan berbau busuk (Schaad
et al 2001). Erwinia carotovora adalah bakteri gram negatif, berbentuk batang hidup soliter atau
berkelompok dalam pasangan atau rantai. Selain bakteri, penyebab kontaminasi pada tomat juga
disebabkan oleh jamur, seperti Aspergillus dan Penicilium (Miskiyah, 2010).
Inokulasi adalah teknik pemindahan mikroba dari medium yang lama ke medium yang baru.
Pemindahan biakan mikroba yang dibiakkan harus dengan hati-hati agar tidak terjadi kontaminasi.
Oleh karena itu, diperlukan metode inokulasi dalam perkembangbiakan mikroorganisme agar
mendapatkan mikroba yang diinginkan (Dwidjoseputro, 2005). Inokulasi merupakan pemindahan
mikroorganisme dari tempat atau sumber asalnya ke media baru yang telah dibuat sebelumnya.
Dimana pada inokulasi masih didapatkan biakan campuran berbagai jenis populasi
mikroorganisme.
Metode – metode yang biasa digunakan dalam inokulasi mikroorganisme ialah metode sebar
(spread plate) dan metode tuang (pour plate). Metode spread plate adalah suatu teknik untuk
menumbuhkan mikroorganisme dengan cara meratakan suspensi pada permukaan agar yang telah
mengeras menggunakan drugalsky agar mikroba tumbuh merata di cawan (Ramadhani, 2014).
Metode pour plate adalah teknik menumbuhkan mikroorganisme pada media agar dengan cara
meletakkan suspensi diantara media (Harley dan Presscot, 2002). Salah-satu proses penting dalam
inokulasi ialah pengenceran bertingkat. Pengenceran bertingkat dilakukan untuk mengurangi
jumlah mikroba sehingga koloni pada cawan tidak berhimpitan. Pengenceran bertingkat
menggunakan larutan fisiologis 0,85%. Mikroba yang diinokulasi ditumbuhkan pada suatu media
sebagai sumber nutrisi.
Media pertumbuhan mikroba adalah suatu bahan yang terdiri atas campuran nutrisi yang
digunakan oleh suatu mikroorganisme untuk tumbuh dan berkembangbiak. Nutrisi yang umumnya
dibutuhkan dalam pertumbuhan mikroba ialah karbohidrat dan protein. Media yang efektif untuk
menumbuhkan bakteri ialah NA karena media NA mengandung banyak nitrogen yang dibutuhkan
oleh bakteri. NA (Nutrient Agar) adalah medium sintesis yang berfungsi untuk enumerasi mikroba
dan medium kultivasi. Komposisinya yaitu intisari peptikum dan sodium klorida 5 gram, ekstrak
daging sapi dan ekstrak khamir 1,5 gram, dan agar 15 gram. Intisari peptikum sebagai sumber
nitrogen, sodium klorida sebagai penyeimbang tekanan osmotik antara medium dan bakteri.
Adapun ekstrak sebagai sumber karbohidrat, vitamin, dan juga garam (Dwidjoseputro 2005).