Download Techniques in basic microbiology

Techniques in basic microbiology
Thanut Amatayakul
Faculty of Agricultural Product Innovation and Technology
Srinakharinwirot University
Outline
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Aseptic techniques
Sterilization
Microscopic study
Microbial Quantification
Media types and preparation
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ASEPTIC CONCEPT AND
TECHNIQUE
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Aseptic
• Processes or activities carried out under sterile condition
• Prevent contamination
– From environment
– To environment
• How???
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Working area and equipment surface disinfection
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Treating surface of working area with disinfectant
Destroying vegetative cells and virus
Endospore is not destroyed
Disinfectant
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70% ethyl alcohol | isopropanol
NaOCl 200 ppm
Fomaldehyde | Glutaraldehyde
Iodine
Phenol
Etc.
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Mechanism-Example
• Ethanol and isopropyl alcohol
– Solubilize lipid in cell wall
– Denature and precipitate protein inside microbial cell
• NaOCl (sodium hypochlorite) 6000 ppm
– oxidises sulphydryl (−SH) and
– disulphide (S−S) bonds in proteins
– Corrosive to stainless steel
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Mechanism
Name
Effective dose
Mechanism
Glutaraldehyde
Phenol
Iodine
Hydrogen peroxide
Ethylene dioxide
Ozone
UV-C
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Effectiveness
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Concentration
Treating time
Temperature
Sensitivity of microbial
Microbial number
Nutrient availability
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Aseptic-Microbial transfer
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Aseptic
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STERILIZATION
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Definition of Sterilization
• Sterilization: processes that are used to eliminate (remove and
kill) microorganism as well as endospore on the surface as well
as in the fluid
• Means
– Heat (dry & wet)
– Chemical (liquid/gas)
– Irradiation
– Filtration
– Pressure
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Filtration-sterilization
• Membrane with pore size 0.22 mm
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Air filter
• Air can be cleaned by passing through HEPA filter (High
Efficiency Particle Air)
• It is mostly used in biological safety cabinet
• Removing particle < 0.3 mm at 99.97%
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Moist heat
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Steam under pressure (high pressure)
121oC for 15 min/135oC for 40 min
Equipment : retort/autoclave
Water boil at 100oC at atmospheric pressure  to achieve > 100oC
need to increase pressure above water
Increase pressure inside autoclave chamber to 103 kPa or 15 lb/in2
 increase temperature to 121oC
To kill spongiform encephalopathies (prions) need higher
temperature or longer time at 121oC
More effective than dry heat
High heat coagulation and denaturation of protein of microbial
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Be careful !!!
It like a boom
ที่มา:
http://www.surge.com.tw/online%
20catalog/online/10.htm
ที่มา : Hogg (2005)
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ที่มา : Hogg (2005)
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Precaution!!!
• It is hot.
• It can be explosive.
• Confirm that pressure come back to atmospheric pressure
before open the vessel.
• Bottle or flask must not be over filled | just 2/3 at most
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Dry heat
• Heat article (metallic) in hot air oven at
160oC for at least 1 hour
• It works by burning the cell
components
• Material should be dried before putting
in the chamber
• The chamber should not be
overcrowded. (air flow)
• The oven should not be open until the
end of sterilizing cycle to prevent
breakage of glassware
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Sterilization by UV (non-ionizing radiation)
• UV ray: 200-280 nm
• 260 nm is the most effective for sterilization
high-pressure mercury vapor lamp
• Induce formation of thymine-thymine dimer inhibiting DNA replication | it do
not kill spore
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ที่มา: http://www.medimagery.com/pathology/bacteria.html
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UV ray
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Do not kill spore
Low penetration
Limit life of UV bulb
Clean surface without dust
Harmful to skin and eye
However, It doesn't penetrate glass, paper or plastic.
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Other vocabulary
• Sanitization: Processes of removing microorganism on the surface of
utensils by either chemical or mechanical means to the level of
safety
• Asepsis : techniques to achieve microbial free environment
• Antiseptic : a chemical mean to eliminate pathogenic
microorganisms from skin
• Bacteriostasis : a condition that bacteria is not multiply, but it is not
death
• Bacteriocidal : a condition that kill or inactivate bacteria
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MICROSCOPIC STUDY
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Light microscope
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Bright field
Dark field
Polarizing
Phase contrast
Fluorescence
ที่มา Benson (2001)
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ที่มา
http://www.visualphotos.com/image/1x3744243/paramecium_bright_field_light_micrograph_of
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Compound light microscope
• Transmission-thin section of sample
• Objective lens: provides a magnified
and inverted image of the specimen
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• Eyepiece lens: adds further
magnification
• Condenser: focuses light onto the
specimen to give optimum illumination
(under stage)
• Magnification: 4x to 100x
• Diaphram: regulate the amount of light
passing through the slide
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Resolution
• It is the resolving power (d)
of lens
• Or the ability to resolve between
Two points a short distance apart
0.61
d
n sin 
 = wavelength of light
n=refractive index of medium between objective lens and cover slide
=aparture angle (or ability of lens to gather light
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n sin  = numerical apparture
ที่มา Hogg (2005)
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Usage
• Assuming that the highest NA. of lens is 1.4 and the lowest visible
wavelength is 400 nm
• Therefore, the resolution is
0.61 400
d
 0.17 mm
1.4
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Magnification
ที่มา
http://www.microscopyu.com/articles/fo
rmulas/formulasmagrange.html
• Total magnification = magnification of objective lens x magnification of
eyepiece
• When the magnification is over the limit of resolution (limit of human eye),
it will give empty magnification
• Useful magnification is approximately 1000 x NA
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Light and medium
• The ability of a microscope objective to capture deviated light rays from a specimen is
dependent upon both the numerical aperture and the medium through which the light
travels.
ที่มา http://www.olympusmicro.com/primer/anatomy/immersion.html
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ที่มา
http://www.olympusmicro.com/primer/anatomy/numaperture
.html
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Lens, oil immerssion, refractive index and Numerical
aperture
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Precaution
ที่มา Benson (2001)
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Using only lint-free cleaning paper
Xylene
Clean lens every time after used
Always start from lower magnification
Use oil immersion with 100x lens
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Stereo microscope
• Use the same principle as Bright field microscope
• With lower magnification (200x)
• Mostly use for examining surface / 3 dimension
ที่มา
ที่มา
http://microscopetalk.files.wordpress.com/2010/07/monilinia_fructico
http://www.microscopyu.com/articles/stereomicroscopy/stereointr
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la_sporangium_stereo2.jpg
o.html
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Microscopes
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Bright field
Phase contrast
Dark field
Fluorescence
Electron microscope
– SEM
– TEM
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Optical microscope
• Light
• Lens
• Bright field, Phase contrast, Dark field
Bright field
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Dark
field
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phase contrast42
Bright field
• Normal compound light microscope
• Use for observing microorganisms
• Microorganisms are needed to be stained to increase contrast
between background and cells
• If not use other kinds of microscope such as dark field or phase
contrast microscope
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Dark field microscope
• A darkfield microscope is used to
examined lived microorganisms that
cannot be seen by ordinary light
microscope method.
• A darkfield microscope uses a darkfield
condenser that contains an opaque disk.
The disk blocks light that would enter the
objective lens directly.
• Only light that is reflected off (turned away
from) the specimen enters the objective
lens. Because there is no direct
background light, the specimen appears
light against a black background-the dark
field
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Phase contrast microscope
• Phase contrast can be used to examine
internal structure of lived microorganisms.
• In phase-contrast microscopy, the specimen is
illuminated by light passing through an annular
(ring shaped) diaphragm. Direct light rays
(unaltered by the specimen) travel a different
path than light rays that are reflected or
diffracted as they pass through the specimen.
These two sets of rays are combined at the
eye. Reflected or diffracted light rays are
indicated in blue; direct rays are red. (Bottom)
Phase-contrast microscopy shows greater
differentiation of internal structures and clearly
shows the pell icle.
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Fluorescence microscope
• Fluorescence: a phomonenone that substance absorb a short
wavelength of light and giving off a longer wavelength of light.
• Some microorganisms fluorescence naturally.
• Some is not. They are needed to be stained with fluorophore or
fluorochrome
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Fluorescence microscope
• Fluorescence antibody technique
• Create antigen (foreign object)
into body and let body
manufacture antibody| remove
and extract antibody
• Similar to bright field but different
at light source (mostly UV) and
wavelength selection device
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Confocal microscope
• This is another type of light
microscope (fluorescence one)
• But instead of illuminating the entire
field, in confocal microscopy, one
plane of a small region of a
specimen is illuminated with a shortwavelength (blue) light which
passes the returned light through
an aperture aligned with the
illuminated region.
• However, it can be used to
reconstruct 3D of cells
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Electron microscope
• Shorter wavelength of electron high resolving power
• SEM & TEM
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EM
• It is suitable for examine
object smaller than 0.2
mm such as internal
organ of microorganism
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New Tool of Fluoresence Microscope
• Super Resolution
Fluorescence
Microscope
Microscopy
• Probe Microscopy
– Magnifies more than 100,000,000 times
– Two types
• Scanning tunneling microscopes
• Atomic force microscopes
Probe microscopy
Figure 4.14
Staining
• Increases contrast and resolution by coloring
specimens with stains/dyes
• Smear of microorganisms (thin film) made prior
to staining
• Microbiological stains contain chromophore
• Acidic dyes stain alkaline structures; more
commonly, basic dyes stain acidic structures
Preparing a specimen for staining
Figure 4.15
Staining
• Simple stains
• Differential stains
– Gram stain
– Acid-fast stain
– Endospore stain
• Special stains
– Negative (capsule) stain
– Flagellar stain
Simple stains
Figure 4.16
The Gram staining procedure
Figure 4.17
Ziehl-Neelsen acid-fast stain
Figure 4.18
Schaeffer-Fulton endospore stain
Figure 4.19
Negative (capsule) stain
Figure 4.20
Flagellar stain
Figure 4.21
DIRECT COUNT
Direct Count
• Through microscope
• Using special equipment “Haemacytometer”
• Counting number of microorganism per field or view under
microscope
• If the volume of sample is known, cell concentration of
microorganism can be calculated.
• However, it is hard to distinguish between live and death
microorganism.
http://www.fisheries.go.th/cf-chan/plankton/hema/hema.htm
http://www.fisheries.go.th/cf-chan/plankton/hema/hema.htm
PLATE COUNT METHOD
Plate Count Method
• Indirect method
• Using culturing medium for growing inoculation cells
• Count only viable cell  as colony forming unit (bacteria) or
CFU
• Need aseptic techniques
• 2 sub categories
– Spread plate
– Pour plate
Problems
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Sometime  Too numereous to count (TNTC)
Serial dilution | mostly 10 fold dilution
1 mL (sample) + 9 mL (sterilie buffer/water)
If dilute too much  Too few to count (TFTC)
Standard ~ 30 – 300 CFU
Calculation
• If you found 47 CFU on petri dish of dilution at 10-5
• How many CFU/mL of original sample?
• The answer is 47 x 105 or 4.7 x 106
Example
• 5 g of contaminated shrimp is tested for bacterial count using
plate count method. 5 g of shrimp is mixed with 100 mL of sterile
buffer. Then, the mixture is diluted (10 fold serial dilution) to 10-4
and spread on culturing plate using 0.1 mL of the mixture. The
result is shown as followed. How many cells of bacteria
contaminate shrimp in CFU/g?
Dilution
CFU
10-1
TNTC
10-2
TNTC
10-3
134
10-4
17
answer
• (134 x 10 x 104 x 100)/5
• 26.8 x 107
• 2.68 x 108
Spread vs Pour plate technique
Spread plate
• Aerobic
• 0.1 mL
• Using spreader to distribute bacteria
on prepared plate
• Plate can be prepared beforehand
(before spreading)
• Serially diluted
• 30-300 CFU (count only colony on
surface)
Pour plate
• Aerobic and Anaerobic
• 1 mL
• Swirling to distribute bacteria
• Serially diluted
• 30-300 CFU (count both colony on
surface and inside midium)
Turbidimetry method
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Indirect measurement of bacterial cells concentration
Measure both live and death cells
Fast at long run
Need to do a calibration curve between bacterial cell count and
absorbance
• Suitable for only microbial in liquid form
TURBIDIMETRIC METHOD
Wavelength ~ 420-660 nm
Must set blank with sterile
Broth
Cell count must be greater
than 107 CFU/mL
Turbidimetric method
• If the OD deviates from a straight line, sample must be diluted
with sterile culture media.
• This technique can be used to rapidly quantify bacteria cell
during fermentation.
• However, limitation of this method is opacity of fermentation
medium such as milk.
FILTRATION
This method can be used to quantify coliform in wate
Cell dry mass (filamentus fungi, yeast)
The cell mass of a very dense cell suspension can be
determined by this technique. In this technique, the
microorganisms are removed from the medium by
filtration and the microorganisms on filters are washed
to remove all extraneous matter, and dried in dessicator
by putting in weighing bottle (previously weighted). The
dried microbial content is then weighted accurately. This
technique is especially useful for measuring the growth
of microfungi. It is time consuming and not very
sensitive. Since bacteria weigh so little, it becomes
necessary to centrifuge several hundred millions of
culture to find out a sufficient quantity to weigh.
การแยกเชื้อจุลินทรี ย ์
การเลี้ยงเชื้อจุลินทรี ย ์
เลี้ยงในสภาพเหมาะสมกับจุลินทรี ยท์ ี่ตอ้ งการศึกษา
• อาหารเลี ้ยงเชื ้อ
– เหลว/แข็ง
• อุณหภูมิ
• ออกซิเจน
• pH
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Media Types
Media
Complex
Defined
Purpose
Grow most heterotrophic organisms
Grow specific heterotrophs and are often mandatory for
chemoautotrophs, photoautotrophs and for microbiological
assays
Selective
Suppress unwanted microbes, or encourage desired microbes
Differential
Distinguish colonies of specific microbes from others
Enrichment
Simlar to selective media but designed to increase the numbers of
desired microorganisms to a detectable level without stimulating
the rest of the bacterial population
Reducing
Growth of obligate anaerobes
http://www.sigmaaldrich.com/analytical-chromatography/microbiology/learning-center/theory/mediapreparation.html
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Media Types
• General-Purpose medium: The medium that is design to grown large spectrum of microorganisms
(nonsynthetic and contain a mixture of nutrients)
– Examples include nutrient agar and broth, brain-heart infusion, and trypticase soy agar (TSA). TSA
contains partially digested milk protein (casein), soybean digest,NaCl, and agar.
• Enriched medium: An enriched medium contains complex organic substances such as blood, serum,
hemoglobin, or special growth factors (specific vitamins, amino acids) that certain species must have
in order to grow.
– Blood agar
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• Selective Medium: A selective medium contains one or
more agents that inhibit the growth of a certain microbe
others (D) and thereby encourages, or selects, microbe D
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• Mannitol salt agar (MSA) contains a concentration of
NaCl (7.5%) that is quite inhibitory to most
genus Staphylococcus, which grows well in this medium
amplified in very mixed samples.
• Bile salts, a component of feces, inhibit mostgrampositive bacteria while permitting many gram-negative
isolating intestinal pathogens (MacConkey agar, eosin
contain bile salts as a selectiveagent.
• Dyes such as methylene blue and crystal violet also
inhibit certain gram-positive bacteria. Other agents that
antimicrobic drugs and acid. Some selective media
to favor the growth of a pathogen that would otherwise
low numbers in a specimen. Selenite and brilliant
isolate Salmonella from feces, and sodium azide is used
water and food.
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• Differential Medium: Differential medium grow several
types of microorganisms and are designed to display
visible differences among those microorganisms.
Differentiation shows up as variations in colony size
or color, in media color changes, or in the formation
of gas bubbles and precipitates
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• For example,
• MacConkey agar contains neutral red, a dye that is yellow when neutral and pink or
yellow when neutral and pink or red when acidic. A common intestinal bacterium such
as Escherichia coli that gives off acid when it metabolizes the lactose in the medium
develops red to pink colonies, and one like Salmonella that does not give off acid
remains its natural color (off-white).
• Spirit blue agar is used to detect the hydrolysis (digestion) of fats by lipase enzyme.
(digestion) of fats by lipase enzyme. Positive hydrolysis is indicated by the dark blue
color that develops in colonies
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• Reducing medium: A reducing medium contains a
substance (thioglycollic acid or cystine) that absorbs
of oxygen in a medium, thus reducing its availability.
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