Materi Seminar Sehari Mikrobiologi "Rapid Microbiological Methods" 05 Sept 2013 IPB Convention Center Bogor

1. Seminar Rapid Microbiological Methods.
International Convention Centre Bogor 5 September 2013
Dra. Syarifah Miftahul El Jannah T.,M.Biomed

2. Latar Belakang
What’s microbiology laboratory activities?
Kualitatatip
isolasi
Identifikasi
Ada atau tidak ada
mikroorganisme
Kuantitatip
Jumlah total
mikroorganisme
Jumlah koloni mo tertentu
Jumlah sel mo tertentu

3. Latar Belakang
5 Fakta Tentang Analisa Mikrobiologi Terkini.
HiGH
COST
????
Lama
Banyak
Alat
Kontaminasi
tinggi
Tenaga
Khusus
Tempat
Khusus

4. Latar Belakang
Keinginan
LOW
COST
????
Cepat
Simpel Kontaminasi
nol
Cara
kerja
general
Lab .
Umum
nya

5. Latar Belakang
PAKAI
TDK YA
????
Mahal Sensitiv
???
Spesifik
???
Harus
ada
pembanding
Regulasi
5 Mitos Tentang Rapid Microbiological
Methods

6. ―Rapid Microbiological
Methods ―
.‖Teknologi terbaru untuk mendeteksi mikroba,
baik untuk identifikasi maupun perhitungan
secara kuantitatif dengan hasil analisa lebih
cepat dibandingkan metoda konvensional ―

7. Kebutuhan Untuk Effisiensi di Semua Lini
Kenapa Kita Perlu
Rapid Microbiological Methods ?
1
2
3
Persyaratan baru di Industri.
Peluang untuk Pengembangan
Berkelanjutan

8. Penurunan Cost Per Analysis
Target Yg Ingin Dicapai dgn
Rapid Microbiological methods
1
2
3
4
Penurunan Lead Time process.
Menjaga Kualitas Produk
Menyesuaikan dengan regulasi terkini.

9. Sample Preparation
Mendapatkan mo yang diinginkan
Bagian yang diambil mewakili
Menghambat mo yang tidak diinginkan
Persyaratan untuk pemeriksaan selanjutnya

10. Sample Preparation
Pre enrichment  Bulyon, Pepton, etc
Enrichment  Selenith, APW, etc
Homogenisasi  Mortar, blender

11. Sample Preparation
Gravimetric Diluter.
This equipment automatically dilutes solids,
semisolids, and liquid samples in dilution 
Penambahan 0.1% pelarut (Manningen and
Fung 1992).
The system, in conjunction with filtration, can
supply sterile diluent and can dispense
presterilized media.

12. Sample Preparation
Stomacher.
When a sample has been weighed and the
appropriate diluent has been added,
dispersion is usually performed by vortexing
or blending in stainless steel blenders
The Stomacher allows for a sample to be dispersed in a sterile plastic
bag by the pummeling action of paddles squashing the sample from
side to side in the bag

13. Sample Preparation
Pulsifier.
uses an oval metal ring surrounding the plastic bag to apply a
high frequency beating action, combined with shock waves and
stirring, to drive microbes into suspension (Fung et al. 1998).
The Pulsifier generates fewer particles than the Stomacher and
thus expands its compatibility to other enumeration and
isolation systems, such as bioluminescence, flow cytometry,
and the polymerase chain reaction (PCR).

14. Media Preparation
Penimbangan
Melarutkan
Penempatan
Sterilisasi
Penyimpanan

15. Media Preparation
Ready Prepared for General Media
 Use prepared media for a variety of applications—e.g., enumeration or
isolation.
 Supply a wide range of solid and liquid media  bulk or individual plate or
tube formats for testing.
 General-purpose media (total viable counts, Staphylococci, coliforms,
yeast, and molds),
 Identification  chromogenic substrates (e.g., Candida albicans,
Escherichia coli, Salmonella spp.) and specific isolation agar (e.g.,
Mycoplasma spp., Helicobacter spp., Neisseria spp.)

16. Media Preparation
Ready Prepared for General Media
The benefit of prepared media :
 Savings in time and resources especially in media preparation and
autoclaving.
 In addition quality assurance benefits related to traceability can be
obtained.
EXPENSIVE VS benefits
One application that is gaining in popularity is the use of pre-prepared
media for environmental monitoring.

17. Media Preparation
PetriFilm, Nissui.
 A system that comprises 2 dry, rehydratable films coated in
nutrients, a cold-soluble gel, and tetrazolium indicator dye for
colony enumeration  incubated for 24 hours at the appropriate
temperature.
 They can be used for air sampling (settle plates), for contact
surface sampling, and also in conjunction with swabs

18. Membrane Filtration
Milliflex System
 The pharmaceutical industry, the method of choice for microbiological
analyses is membrane filtration  the Microfil™ system uses pre-packed
0.45 μ m membranes with a patented vacuum support and disposable funnel.
 An extension to Microfil is the Milliflex™ 100 system, in which the
disposable funnel and membrane are combined as a single pre-sterilized unit.
The combined unit is placed on the Millipore vacuum system, the sample is
filtered and the filter assembly then snapped into a prefilled medium cassette,
and the membrane is then incubated  Water system (purified water for
injection) sampling is a critical process requiring trained samplers.

19. Membrane Filtration
Milliflex System
The Milliflex P system minimizes extraneous contamination.
This closed system comprises a 0.45 μ m filter membrane within a cassette,
which can be directly attached to a sanitary design sampling port, and water
samples are filtered directly.
After the sampling, the filtration unit is directly coupled to a prefilled media
cassette and incubated

20. Membrane Filtration
Steritest System
 Membrane-based device that is attaining significant use in the
pharmaceutical industry.
Steritest is a closed method for sterility testing, designed to be compatible
with a range of dosage forms and packaging types—e.g., bottles, vials,
ampoules, syringes, collapsible bags, antibiotics, creams and ointments,
aerosols, and liquids.
The Steritest reduces the risk of false positives by minimizing technician
manipulations and guarding against adventitious contamination.
 However, it still requires 14 of days incubation.

21. Spesific Organism Isolation
Define Substrate Technology .
involves the linking of an enzyme substrate to a colorless
chemical indicator such that when the substrate is used a change
in color or fluorescence is observed.
 Incorporating O-nitrophenyl-B-D-galactopyronoside (ONPG) to
detect coliforms and 4-methyl-umbelliferone glucuronide (MUG) to
detect E. Coli (using ultraviolet light).

22. Spesific Organism Isolation
Colilert, Coliquick
 Commercially available media have been developed—e.g.,
Colilert™ and Coliquick™ (IDEXX Labortories, United States), and
Colifast® (Colifast AS, Norway)—based upon ONPG and MUG.
 Arange of alternative agars incorporating different dyes,
substrates, and selective agents are available—e.g., BBL
CHROMagar™ (Becton Dickenson, France),

23. Spesific Organism Isolation
Chromogenic Media
Detecting specific enzyme activities using substrates that release
coloured or fluorescent components on hydrolysis allowing the
specific presumptive identification
ChromAgar, HiChrome, Cromocult Coli Agar.
Chromocult™ (Merck, Germany), BCM™ agars (Biosynth International Inc.,
United States), M-Coli blue (Camlab, United Kingdom), Rainbow® agars
(Biolog, Inc., United States), and Simplate (IDEXX).

24. JENIS MEDIA PENGUJI

25. Harlequin™ Salmonella ABC HAL001
• Dual Chromogens are used to isolate Salmonella spp
• First substrate is metabolised by most
Enterobacteriaceae, giving rise to black colonies
• Salmonella spp will also hydrolyse the second
substrate producing very distinguished green colonies

26. Harlequin™ Salmonella ABC HAL001

27. Immuno-magnetic separation (IMS)
Isolation and separation of specific microorganisms by use of
selective media and enrichment broths may be enhanced by the use of
immuno-magnetic separation (IMS).
IMS is a non-growth-related procedure that utilizes super-
paramagnetic beads,
linked to antibodies or lectins, that bind to specific organisms these
beads will attach to target organisms, and after the specified incubation
period (10–60 minutes) an external magnet is applied to the
suspension, and the beads are segregated from the rest of the sample
matrix.

28. Immuno-magnetic separation (IMS)
IMS can be linked to other rapid microbiological technologies, such as
impedance, immunoassays, and DNA hybridization
and amplification technologies. Isolating specific organ isms from
competing flora and any inhibiting substances should
reduce the time to detection. It must be remembered that although
IMS will isolate stressed organisms, it will also recover ead organisms
that may cause false positives in polymerase chain reaction (PCR)
systems.

29. Anaerob
Oxyrase.
 Pharmaceutical companies perform tests for anaerobic organisms to
varying degrees, from non-routine environmentalnmonitoring sampling
to analyses on every finished product batch.
 Oxyrase® (Oxyrase, United States) is a membrane fraction of E. coli
that can convert oxygen to water, thereby reducing oxygen tension and
creating anaerobic conditions. Such anaerobic conditions can be
maintained in both liquid and solid media.

30. Anaerob
Oxyrase.
. When used in conjunction with the Oxydish™ (Oxyrase, Inc.), an
anaerobic headspace can be maintained, allowing surface colony
growth of anaerobic organisms (Gannon and Thurston 1996).
The use of Oxyrase negates the need for anaerobic chambers, inert
gas cylinders, or other devices. Oxyrase has also been found to
stimulate the growth of injured and uninjured, facultative, and anaerobic
organisms (Adler and Spady 1997). Oxyrase has already been used in
media fill simulations during asceptic filling line validations.

31. Immunological Methods
Enzym Linked Immunosorbent Assay.
 Enzyme immunoassays (EIAs) /enzyme linked immunosorbant assays
(ELISAs) / latex agglutination  in the food industry for rapid screening or
confirmation of pathogenic organisms (Salmonella species, E. coli O157,
Listeria, and Staphylococcus aureus.)
 Immunological methods are useful for screening and detecting specific
microorganisms and their toxins.
 They are quicker than conventional methods, especially if used in
conjunction with immunoconcentration techniques, such as IMS.

32. Immunological Methods
Enzym Linked Immunosorbent Assay.
 Preenrichment is commonly a requirement, because immunological
methods require=104 cells/mL to ensure detection.
 With these requirements, assay times vary from 24 to 52 hours for
specific bacteria and 4 to 24 hours for microbial toxins.
 The major drawbacks are false positives due to nonspecific binding and
false negatives in that dead cells are also likely to be detected.

33. Electrical Methods
Impedance ( Bactometer ).
 The point at which changes in electrical measurements are recognized as
a result of microbial activity is known as the time to detection (TTD).
 This parameter is the time taken at which the number of microorganisms
reaches a critical mass that produces a significant change in the electrical
activity of the media.
This critical mass is usually around 106 cells/mL (Cady et al. 1978).
 Time to detection is directly correlated to the number of organisms in the
original sample and also the growth rate of these organisms is known as the
time to detection (TTD)  Kurva pertumbuhan

34. Electrical Methods
Impedance ( Bactometer ).
 The measured TTD from the sample can give a direct estimate of the
initial sample population.
.The advantage of these systems is that they are automated, are simple
to use, and have a very high throughput of samples.
 They can undertake a large number of experiments at any one time,
and individual samples can be accessed without disturbing the others.
 Cost per sample is very low.

35. Electrical Methods
Impedance ( Bactometer ).
 qualitative screening test for pharmaceutical raw materials,
intermediates, finished products, and even environmental samples.
 An application for which electrical technology is ideally suited is the
evaluation of antibiotic resistance and, in particular, preservative efficacy.
 Connolly et al. (1994) demonstrated that impedance can be used for
preservative efficacy testing (PET) of pharmaceuticals and cosmetics.

36. Electrical Methods
Bioluminescence ATP ( Biotrace, HyLite,
kikkoman ).
 All living cells possess adenosine triphosphate (ATP) as the main chemical
energy store.
 In the presence of the substrate Dluciferin, oxygen, and magnesium ions, the
enzyme luciferase will utilize the energy from ATP to oxidize D-luciferin and
produce light.
 The amount of light or bioluminescence produced can be measured by
sensitive luminometers and is proportional to the amount of ATP present in the

37. Electrical Methods
Bioluminescence ATP ( Biotrace, HyLite,
kikkoman ).
 All living cells possess adenosine triphosphate (ATP) as the main chemical
energy store.
 In the presence of the substrate Dluciferin, oxygen, and magnesium ions, the
enzyme luciferase will utilize the energy from ATP to oxidize D-luciferin and
produce light.
ATP + D-luciferins + O2-luciferase  AMP + Oxyluciferase + CO2 + ppi + LIGHT
Mg

38. Electrical Methods
Bioluminescence ATP ( Biotrace, HyLite,
kikkoman ).
 The amount of light or bioluminescence produced can be measured by
sensitive luminometers and is proportional to the amount of ATP present in the
sample.
 The emitted light is usually expressed as relative light units (RLUs).
ATP bioluminescence offers many advantages in terms of speed to result and
ease of use sensitivity—requiring 103–104 organisms for detection

39. Electrical Methods
Bioluminescence ATP ( Biotrace, HyLite,
kikkoman ).
 Samples may also contain endogenous, nonmicrobial ATP, providing false
positive reactions
 disadvantage is that the release of ATP from the cells is disruptive, and
therefore further evaluation of any organisms recovered from the sample canot
be performed
 Useful in the food industry, The presence of soil containing microbial and/or
nonmicrobial ATP is an indicator of hygiene testing.

40. Electrical Methods
Bioluminescence ATP ( Biotrace, HyLite,
kikkoman ).
 Potentially possible to perform real time measurements for water quality, air
monitoring, direct liquid product monitoring, surface environmental monitoring,
and also filterable materials—e.g., raw materials, intermediates, finished
products.
 Sensitivity of ATP bioluminescence still remains at 103 organisms, the reason
for a growth or pre-enrichment phase.

41. Electrical Methods
Bioluminescence ATP ( Biotrace, HyLite,
kikkoman ).
 Highlighted that sensitivity of the direct ATP bioluminescence reaction could
be increased by coupling to adenylate kinase (AK) activity. AK is an intracellular
enzyme, present in all living things, that catalyzes the reaction
 ATP amplification can occur in the cell through the use of AK. reactions
should allow for the detection of a single CFU within 25 minutes.
2 ADP  AMP + ATP

42. Electrical Methods
Laser Scaning Cytometri ( ScanRDI, DCount
).
 Combines membrane filtration, direct viability staining
(epifluorescence), and laser scanning.
 Chemunex SA (France) has developed the ScanRDI®. This system is
only for filterable materials but has a sensitivity of 1 CFU per sample with
a result in less than 2 hours.
 Sample  filtered  labeled (the passive diffusion of a
nonfluorescent substrate derived from fluorescein (Fluorassure®).  and
then laser scanned.

43. Electrical Methods
Laser Scaning Cytometri ( ScanRDI, DCount
).
 The fluorescein substrate is cleaved by esterase activity in the cell
cytoplasm, and its retention within the cell is a function of the organisms
cell membrane.
 Only viable organisms are detected, on the basis of metabolic activity
and an intact plasma membrane.
 Benefits of the technology are its speed (result within 2 hours), its
sensitivity, and the absence of requirementsfor growth, avoiding pre-
enrichment or selective processes for fastidious or stressed
microorganisms.

44. Moleculer Based Technology
Genotyping
ChemoTaxonomy.
Electrophoretic Protein Typing.

45. • Restriction Endonuclear Analysis.
• Plasmid Typing.
• Ribotyping.
• PTGE Typing.
• PCR Base Typing System.
• Identification with primers and probes.
Genotyping

46. • Mass Spectrometry and Pyrolisis.
• FTIR.
• Gas Chromatography.
• Microbial Identification System.
ChemoTaxonomy

47. • PCR Base Typing System.
Genotyping
a biochemical technology in molecular biology to
amplify a single or a few copies of a piece of DNA
across several orders of magnitude, generating
thousands to millions of copies of a particular DNA
sequence

48. (1) Denaturing at 94–96 °C.
(2) Annealing at ~65 °C
(3) Elongation at 72 °C.
Four cycles are shown here.
The blue lines represent the
DNA template to which
primers (red arrows) anneal
that are extended by the DNA
polymerase (light green
circles), to give shorter DNA
products (green lines), which
themselves are used as
templates as PCR progresses.

49. Counting Procedure
Hydrophobic Grid Membrane Filtration.
Automatic Colony Counter

50. Identification System
Micro Id.
API
Enterotube.
Crystal.

52. BANDINGKAN......................

53. Top-Ten Attributes of an Ideal Rapid Test
(Daniel Y. C. Fung, Professor of Food Science, Kansas State University,
Manhattan)
that many factors contribute to making a rapid test ideal to use.
1. Accurate for the intended purposes. It considers sensitivity, minimal detectable limits,
specificity of test system, versatility, potential applications, and comparison to reference
methods.
2. Speedy and productive. It includes time in obtaining results and the number of
samples processed per run, per hour, and per day.
3. Cost effective. The initial, per-test, reagent, and labor costs are reasonable.

54. 4. Acceptable. It is acceptable by the scientific community and regulatory agencies.
5. Simple to operate. The test takes into consideration sample preparation, operation of
test equipment, and computer versatility.
6. Amenable to training. Location (on or off site), length of time, and qualifications of
operator are all factors.
7. User-friendly reagents. Preparation, stability, availability, and consistency of reagents
need to be considered.
8. Great company reputation.
9. Exceptional technical service. Speed, availability, cost, and scope all count.
10. Optimum utility and space requirements.

55. If you think that you are
beaten, you are;
If you think you dare not,
you don’t;
If you’d like to win, but think
that you can’t,
It s almost a cinch you
won’t.
If you think that you’ll lose,
you’re lost,
For out in the world we find
Success begins with a