1. Techniques in basic microbiology
Thanut Amatayakul
Faculty of Agricultural Product Innovation and Technology
Srinakharinwirot University

2. Outline Aseptic techniques Sterilization Microscopic study
Microbial Quantification
Media types and preparation
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3. Aseptic concept and technique
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4. Aseptic Processes or activities carried out under sterile condition
Prevent contamination
From environment
To environment
How???
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5. Working area and equipment surface disinfection
Treating surface of working area with disinfectant
Destroying vegetative cells and virus
Endospore is not destroyed
Disinfectant
70% ethyl alcohol | isopropanol
NaOCl 200 ppm
Fomaldehyde | Glutaraldehyde
Iodine
Phenol
Etc.
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6. 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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7. Mechanism Name Effective dose Mechanism Glutaraldehyde Phenol Iodine
Hydrogen peroxide
Ethylene dioxide
Ozone
UV-C
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10. Effectiveness Concentration Treating time Temperature
Sensitivity of microbial
Microbial number
Nutrient availability
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11. Aseptic-Microbial transfer
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12. Aseptic
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13. Sterilization
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14. 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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16. Filtration-sterilization
Membrane with pore size 0.22 mm
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17. 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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19. Moist heat 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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20. Be careful !!! It like a boom ที่มา : Hogg (2005)
ที่มา:
ที่มา : Hogg (2005)
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21. ที่มา : Hogg (2005)
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22. 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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23. 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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24. Sterilization by UV (non-ionizing radiation)
UV ray: nm
260 nm is the most effective for sterilization
Induce formation of thymine-thymine dimer inhibiting DNA replication | it do not kill spore
high-pressure mercury vapor lamp
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25. ที่มา: http://www.medimagery.com/pathology/bacteria.html
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26. UV ray 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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27. 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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28. Microscopic study
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29. Light microscope Bright field Dark field Polarizing Phase contrast
Fluorescence
ที่มา Benson (2001)
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30. ที่มา
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31. Compound light microscope
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
Transmission-thin section of sample
Objective lens: provides a magnified and inverted image of the specimen
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32. Resolution It is the resolving power (d) of lens
Or the ability to resolve between
Two points a short distance apart
ที่มา Hogg (2005)
 = wavelength of light
n=refractive index of medium between objective lens and cover slide
=aparture angle (or ability of lens to gather light
n sin  = numerical apparture
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33. Usage
Assuming that the highest NA. of lens is 1.4 and the lowest visible wavelength is 400 nm
Therefore, the resolution is
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34. Magnification
ที่มา
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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35. 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
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36. ที่มา http://www.olympusmicro.com/primer/anatomy/numaperture.html
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37. Lens, oil immerssion, refractive index and Numerical aperture
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38. Precaution Using only lint-free cleaning paper Xylene
Clean lens every time after used
Always start from lower magnification
Use oil immersion with 100x lens
ที่มา Benson (2001)
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39. Stereo microscope Use the same principle as Bright field microscope
With lower magnification (200x)
Mostly use for examining surface / 3 dimension
ที่มา
ที่มา
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41. Microscopes Bright field Phase contrast Dark field Fluorescence
Electron microscope
SEM
TEM
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42. Optical microscope Light Lens Bright field, Phase contrast, Dark field
Bright field Dark field phase contrast
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43. 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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44. 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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45. 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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46. 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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47. 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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48. 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 short-wavelength (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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49. Electron microscope
Shorter wavelength of electron high resolving power
SEM & TEM
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50. EM
It is suitable for examine object smaller than 0.2 mm such as internal organ of microorganism
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52. New Tool of Fluoresence Microscope
Super Resolution Fluorescence Microscope

53. Microscopy Probe Microscopy Magnifies more than 100,000,000 times
Two types
Scanning tunneling microscopes
Atomic force microscopes

54. Probe microscopy
Figure 4.14

55. 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

56. Preparing a specimen for staining
Figure 4.15

57. Staining Simple stains Differential stains Special stains Gram stain
Acid-fast stain
Endospore stain
Special stains
Negative (capsule) stain
Flagellar stain

58. Simple stains
Figure 4.16

59. The Gram staining procedure
Figure 4.17

60. Ziehl-Neelsen acid-fast stain
Figure 4.18

61. Schaeffer-Fulton endospore stain
Figure 4.19

62. Negative (capsule) stain
Figure 4.20

63. Flagellar stain
Figure 4.21

64. Direct count

65. 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.

72. Plate count method

73. 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

74. Problems 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

77. 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

78. 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
10-3
134
10-4 17

79. answer
(134 x 10 x 104 x 100)/5
26.8 x 107
2.68 x 108

80. Spread vs Pour plate technique
Spread plate
Pour plate
Aerobic
0.1 mL
Using spreader to distribute bacteria on prepared plate
Plate can be prepared beforehand (before spreading)
Serially diluted
CFU (count only colony on surface)
Aerobic and Anaerobic
1 mL
Swirling to distribute bacteria
Serially diluted
CFU (count both colony on surface and inside midium)

82. Turbidimetry method
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

83. Turbidimetric method

84. Wavelength ~ nm
Must set blank with sterile
Broth
Cell count must be greater than 107 CFU/mL

86. 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.

87. Filtration

88. This method can be used to quantify coliform in water

89. 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.

90. การแยกเชื้อจุลินทรีย์

91. การเลี้ยงเชื้อจุลินทรีย์ เลี้ยงในสภาพเหมาะสมกับจุลินทรีย์ที่ต้องการศึกษา
อาหารเลี้ยงเชื้อ
เหลว/แข็ง
อุณหภูมิ
ออกซิเจน pH
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92. Media Types Media Purpose Complex Grow most heterotrophic organisms
Defined
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
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93. 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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94. Selective Medium: A selective medium contains one or more agents that inhibit the growth of a certain microbe or microbes (A, B, C) but not others (D) and thereby encourages, or selects, microbe D and allows it to grow.
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95. Mannitol salt agar (MSA) contains a concentration of NaCl (7
Mannitol salt agar (MSA) contains a concentration of NaCl (7.5%) that is quite inhibitory to most humanpathogens. One exception is the genus Staphylococcus, which grows well in this medium and consequently can be amplified in very mixed samples.
Bile salts, a component of feces, inhibit mostgram-positive bacteria while permitting many gram-negative rods to grow. Media for isolating intestinal pathogens (MacConkey agar, eosin methylene blue [EMB] agar) contain bile salts as a selectiveagent.
Dyes such as methylene blue and crystal violet also inhibit certain gram-positive bacteria. Other agents that have selective properties are antimicrobic drugs and acid. Some selective media contain strongly inhibitory agents to favor the growth of a pathogen that would otherwise be overlooked because of its low numbers in a specimen. Selenite and brilliant green dye are used in media to isolate Salmonella from feces, and sodium azide is used to isolate enterococci from water and food.
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96. 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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97. For example,
MacConkey agar contains neutral red, a dye that is 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. Positive hydrolysis is indicated by the dark blue color that develops in colonies
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98. Reducing medium: A reducing medium contains a substance (thioglycollic acid or cystine) that absorbs oxygen or slows the penetration of oxygen in a medium, thus reducing its availability.
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